Genetic markers are employed in  genealogical DNA testing  for  genetic genealogy  to determine  genetic distance  between individuals or populations. Uniparental markers (on  mitochondrial  or  Y chromosomal  DNA) are studied for assessing maternal or paternal  lineages .  Autosomal  markers are used for all ancestry.


Genetic markers have to be easily identifiable, associated with a specific  locus , and highly  polymorphic , because  homozygotes  do not provide any information. Detection of the marker can be direct by RNA sequencing, or indirect using  allozymes .


Some of the methods used to study the  genome  or  phylogenetics  are RFLP, AFLP, RAPD, SSR. They can be used to create genetic maps of whatever organism is being studied.


There was a debate over what the transmissible agent of  CTVT  ( canine transmissible venereal tumor ) was. Many researchers hypothesized that virus like particles were responsible for transforming the cell, while others thought that the cell itself was able to infect other canines as an  allograft . With the aid of genetic markers, researchers were able to provide conclusive evidence that the cancerous tumor cell evolved into a transmissible parasite. Furthermore, molecular genetic markers were used to resolve the issue of natural transmission, the breed of origin ( phylogenetics ), and the age of the canine tumor. [ 4 ]


Genetic markers have also been used to measure the genomic response to selection in livestock. Natural and artificial selection leads to a change in the genetic makeup of the cell. The presence of different alleles due to a distorted segregation at the genetic markers is indicative of the difference between selected and non-selected livestock. [ 5 ]


Media related to  Genetic markers  at Wikimedia Commons


Genetic testing , also known as  DNA testing , is used to identify changes in  DNA  sequence or  chromosome  structure. Genetic testing can also include measuring the results of genetic changes, such as  RNA  analysis as an output of  gene expression , or through  biochemical analysis  to measure specific  protein  output. [ 1 ]  In a medical setting, genetic testing can be used to diagnose or rule out suspected  genetic disorders ,  predict risks  for specific conditions, or gain information that can be used to customize medical treatments based on an individual's genetic makeup. [ 1 ]  Genetic testing can also be used to determine biological relatives, such as a child's  biological parentage  (genetic mother and father) through  DNA paternity testing , [ 2 ]  or be used to broadly predict an individual's  ancestry . [ 3 ]  Genetic testing of plants and animals can be used for similar reasons as in humans (e.g. to assess relatedness/ancestry or predict/diagnose genetic disorders), [ 4 ]  to gain information used for  selective breeding , [ 5 ]  or for efforts to boost  genetic diversity  in endangered populations. [ 6 ]


The variety of genetic tests has expanded throughout the years. Early forms of genetic testing which began in the 1950s involved counting the number of chromosomes per cell. Deviations from the expected number of chromosomes (46 in humans) could lead to a diagnosis of certain genetic conditions such as trisomy 21 ( Down syndrome ) or monosomy X ( Turner syndrome ). [ 7 ]   In the 1970s, a method to stain specific regions of chromosomes, called  chromosome banding , was developed that allowed more detailed  analysis  of chromosome structure and diagnosis of genetic disorders that involved large structural rearrangements. [ 8 ]  In addition to analyzing whole chromosomes ( cytogenetics ), genetic testing has expanded to include the fields of  molecular genetics  and  genomics  which can identify changes at the level of individual genes, parts of genes, or even single  nucleotide  "letters" of DNA sequence. [ 7 ]   According to the  National Institutes of Health , there are tests available for more than 2,000 genetic conditions, [ 9 ]  and one study estimated that as of 2018 there were more than 68,000 genetic tests on the market. [ 10 ]


Genetic testing is "the analysis of  chromosomes  ( DNA ), proteins, and certain  metabolites  in order to detect heritable disease-related  genotypes ,  mutations ,  phenotypes , or  karyotypes  for clinical purposes." [ 11 ]  It can provide information about a  person 's genes and chromosomes throughout life.


Genetic testing is often done as part of a genetic consultation and as of mid-2008 there were more than 1,200 clinically applicable genetic tests available. [ 23 ]  Once a person decides to proceed with genetic testing, a medical geneticist, genetic counselor, primary care doctor, or specialist can order the test after obtaining  informed consent . [ citation needed ]


Genetic tests are performed on a sample of  blood ,  hair ,  skin ,  amniotic fluid  (the fluid that surrounds a fetus during pregnancy), or other tissue. For example, a  medical procedure  called a  buccal smear  uses a small brush or cotton swab to collect a sample of cells from the inside surface of the cheek. Alternatively, a small amount of saline  mouthwash  may be swished in the mouth to collect the cells. The sample is sent to a laboratory where technicians look for specific changes in chromosomes, DNA, or proteins, depending on the suspected disorders, often using  DNA sequencing . The laboratory reports the test results in writing to a person's doctor or genetic counselor. [ citation needed ]


Routine  newborn  screening tests are done on a small blood sample obtained by pricking the baby's heel with a  lancet .


The physical risks associated with most genetic tests are very small, particularly for those tests that require only a blood sample or buccal smear (a procedure that samples cells from the inside surface of the cheek). The procedures used for prenatal testing carry a small but non-negligible risk of losing the pregnancy (miscarriage) because they require a sample of amniotic fluid or tissue from around the fetus. [ 24 ]


Many of the risks associated with genetic testing involve the emotional, social, or financial consequences of the test results. People may feel angry, depressed, anxious, or guilty about their results. The potential negative impact of genetic testing has led to an increasing recognition of a "right not to know". [ 25 ]  In some cases, genetic testing creates tension within a family because the results can reveal information about other family members in addition to the person who is tested. [ 26 ]  The possibility of  genetic discrimination  in employment or insurance is also a concern. Some individuals avoid genetic testing out of fear it will affect their ability to purchase insurance or find a job. [ 27 ]  Health insurers do not currently require applicants for coverage to undergo genetic testing, and when insurers encounter genetic information, it is subject to the same confidentiality protections as any other sensitive health information. [ 28 ]  In the United States, the use of genetic information is governed by the  Genetic Information Nondiscrimination Act  (GINA) (see discussion below in the section on government regulation).


Genetic testing can provide only limited information about an inherited condition. The test often can't determine if a person will show symptoms of a disorder, how severe the symptoms will be, or whether the disorder will progress over time. Another major limitation is the lack of treatment strategies for many genetic disorders once they are diagnosed. [ 24 ]


Another limitation to genetic testing for a hereditary linked cancer, is the variants of unknown clinical significance. Because the human genome has over 22,000 genes, there are 3.5 million variants in the average person's genome. These variants of unknown clinical significance means there is a change in the DNA sequence, however the increase for cancer is unclear because it is unknown if the change affects the gene's function. [ 29 ]


A genetics professional can explain in detail the benefits, risks, and limitations of a particular test. It is important that any person who is considering genetic testing understand and weigh these factors before making a decision. [ 24 ]


Other risks include  incidental findings —a discovery of some possible problem found while looking for something else. [ 30 ]  In 2013 the American College of Medical Genetics and Genomics (ACMG) recommended that certain genes always be included any time a genomic sequencing was done, and that labs should report the results. [ 31 ]


DNA studies have been criticised for a range of methodological problems and providing misleading, interpretations on racial classifications. [ 32 ] [ 33 ] [ 34 ] [ 35 ] [ 36 ]


Direct-to-consumer  (DTC) genetic testing (also called at-home genetic testing) is a type of genetic test that is accessible directly to the consumer without having to go through a health care professional. Usually, to obtain a genetic test, health care professionals such as physicians, nurse practitioners, or  genetic counselors  acquire their patient's permission and then order the desired test, which may or may not be covered by health insurance. DTC genetic tests, however, allow consumers to bypass this process and purchase DNA tests themselves. DTC genetic testing can entail primarily genealogical/ancestry-related information, health and trait-related information, or both. [ 37 ]  Genetic testing has been taken on by private companies, such as  23andMe ,  Ancestry.com , and  Family Tree DNA . These companies will send the consumer a kit at their home address, with which they will provide a saliva sample for their lab to analyze. The company will then send back the consumer's results in a few weeks, which is a breakdown of their ancestral heritage and possible health risks that accompany it. [ 38 ]


There are a variety of DTC genetic tests, ranging from tests for breast cancer alleles to mutations linked to  cystic fibrosis . Possible benefits of DTC genetic testing are the accessibility of tests to consumers, promotion of proactive healthcare, and the  privacy of genetic information . Possible additional risks of DTC genetic testing are the lack of governmental regulation, the potential misinterpretation of genetic information, issues related to testing minors,  privacy of data , and downstream expenses for the public health care system. [ 39 ]  In the United States, most DTC genetic test kits are not reviewed by the  Food and Drug Administration (FDA) , with the exception of a few tests offered by the company  23andMe . [ 40 ]  As of 2019, the tests that have received marketing authorization by the FDA include 23andMe's genetic health risk reports for select variants of  BRCA1/BRCA2 , [ 41 ]   pharmacogenetic  reports that test for selected variants associated with metabolism of certain pharmaceutical compounds, a carrier screening test for  Bloom syndrome , and genetic health risk reports for a handful of other medical conditions, such as  celiac disease  and  late-onset Alzheimer's . [ 40 ]


DTC genetic testing has been controversial due to outspoken opposition within the medical community. Critics of DTC genetic testing argue against the risks involved in several steps of the testing process, such as the unregulated advertising and  marketing claims , the potential reselling of genetic data to third parties, and the overall lack of governmental oversight. [ 42 ] [ 43 ] [ 44 ] [ 45 ]


DTC genetic testing involves many of the same risks associated with any genetic test. One of the more obvious and dangerous of these is the possibility of misreading of test results. Without professional guidance, consumers can potentially misinterpret genetic information, causing them to be deluded about their personal health.


Some advertising for DTC genetic testing has been criticized as conveying an exaggerated and inaccurate message about the connection between genetic information and disease risk, utilizing emotions as a selling factor. An advertisement for a  BRCA -predictive genetic test for breast cancer stated: "There is no stronger antidote for fear than information." [ 46 ]  Apart from rare diseases that are directly caused by specific, single-gene mutation, diseases "have complicated, multiple genetic links that interact strongly with personal environment, lifestyle, and behavior." [ 47 ]


Ancestry.com , a company providing DTC DNA tests for  genealogy  purposes, has reportedly allowed the  warrantless search  of their database by police investigating a murder. [ 48 ]  The warrantless search led to a search warrant to force the gathering of a DNA sample from a New Orleans filmmaker; however he turned out not to be a match for the suspected killer. [ 49 ]


As part of its healthcare system, Estonia is offering all of its residents genome-wide genotyping. This will be translated into personalized reports for use in everyday medical practice via the national e-health portal. [ 50 ]


The aim is to minimise health problems by warning participants most at risk of conditions such as cardiovascular disease and diabetes. It is also hoped that participants who are given early warnings will adopt  healthier lifestyles  or take  preventative drugs . [ 51 ]


In 2005, National Geographic launched the "Genographic Project", which was a fifteen-year project that was discontinued in 2020. Over one million people participated in the DNA sampling from more than 140 countries, which made the project the largest of its kind ever conducted. [ 52 ]  The project asked for DNA samples from indigenous people as well as the general public, which spurred political controversy among some indigenous groups, leading to the coining of the term "biocolonialism". [ 53 ]


With regard to genetic testing and information in general, legislation in the United States called the  Genetic Information Nondiscrimination Act  prohibits group health plans and health insurers from denying coverage to a healthy person or charging that person higher premiums based solely on a  genetic predisposition  to developing a disease in the future. The legislation also bars employers from using genetic information when making  hiring ,  firing , job placement, or  promotion  decisions. [ 54 ]
Although GINA protects against genetic discrimination, Section 210 of the law states that once the disease has manifested, employers can use the medical information and not be in violation of the law, even if the condition has a genetic basis. [ 55 ]  The legislation, the first of its kind in the United States, [ 56 ]  was passed by the  United States Senate  on April 24, 2008, on a vote of 95–0, and was signed into law by President  George W. Bush  on May 21, 2008. [ 57 ] [ 58 ]  It went into effect on November 21, 2009.


In June 2013 the US Supreme Court issued two rulings on human genetics. The Court struck down patents on human genes, opening up competition in the field of genetic testing. [ 59 ]  The Supreme Court also ruled that police were allowed to collect DNA from people arrested for serious offenses. [ 60 ]


Effective as of 25 May 2018, companies that process genetic data must abide by the  General Data Protection Regulation  (GDPR). [ 61 ] [ 62 ]  The GDPR is a set of rules/regulations that helps an individual take control of their data that is collected, used, and stored digitally or in a structured filing system on paper, and restricts a company's use of personal data. [ 62 ]  The regulation also applies to companies that offer products/services outside the EU. [ 62 ]


Genetic testing in Germany is governed by the Genetic Diagnostics Act (GenDG), [ 63 ]  which mandates that health-related genetic tests can only be carried out under medical supervision to ensure the proper interpretation of results and informed decision-making. The law emphasizes genetic counseling and informed consent, protecting individuals from potential misuse or misunderstanding of their genetic data.


The legal status of genetic testing in France is regulated under strict privacy and data protection laws, including the Bioethics Law. [ 64 ]  Direct-to-consumer (DTC) genetic tests, especially those for health-related purposes, are prohibited unless conducted with medical oversight to ensure informed consent and appropriate counseling. [ 65 ]  This is due to concerns about the potential misuse of genetic data and privacy violations. While health-related genetic testing is allowed within a medical context, tests for non-medical purposes, such as ancestry or personal traits, also face legal restrictions, particularly regarding consumer access.


Russian law [ 66 ]  provides that the processing of special categories of personal data relating to race, nationality, political views, religious or philosophical beliefs, health status, intimate life is allowed if it is necessary in connection with the implementation of international agreements of the Russian Federation on readmission and is carried out in accordance with the legislation of the Russian Federation on citizenship of the Russian Federation. Information characterizing the physiological and biological characteristics of a person, on the basis of which it is possible to establish his identity (biometric personal data), can be processed without the consent of the subject of personal data in connection with the implementation of international agreements of the Russian Federation on readmission, administration of justice and execution of judicial acts, compulsory state fingerprinting registration, as well as in cases stipulated by the legislation of the Russian Federation on defense, security, anti-terrorism, transport security, anti-corruption, operational investigative activities, public service, as well as in cases stipulated by the criminal-executive legislation of Russia, the legislation of Russia on the procedure for leaving the Russian Federation and entering the Russian Federation, citizenship of the Russian Federation and notaries.


Within the framework of this program, it is also planned to include the peoples of neighboring countries, which are the main source of migration, into the genogeographic study on the basis of existing collections. [ 67 ]


By the end of 2021, the UAE Genome Project will be in full swing, as part of the National Innovation Strategy, establishing strategic partnerships with top medical research centers, and making sustainable investments in healthcare services. The project aims to prevent genetic diseases through the use of genetic sciences and innovative modern techniques related to profiling and genetic sequencing, in order to identify the genetic footprint and prevent the most prevalent diseases in the country, such as obesity, diabetes, hypertension, cancer, and asthma. It aims to achieve personalized treatment for each patient based on genetic factors. Additionally, a study by Khalifa University has identified, for the first time, four genetic markers associated with type 2 diabetes among UAE citizens. [ 68 ]


The Israeli  Knesset  passed the Genetic Information Law in 2000, becoming one of the first countries to establish a regulatory framework for the conducting of genetic testing and genetic counseling and for the handling and use identified genetic information. Under the law, genetic tests must be done in labs accredited by the  Ministry of Health ; however, genetic tests may be conducted outside Israel. The law also forbids discrimination for employment or insurance purposes based on genetic test results. Finally, the law takes a strict approach to genetic testing on minors, which is permitted only for the purpose of finding a genetic match with someone ill for the sake of medical treatment, or to see whether the minor carries a gene related to an illness that can be prevented or postponed. [ 69 ] [ 70 ]


Under the Genetic Information Law as of 2019, commercial DNA tests are not permitted to be sold directly to the public, but can be obtained with a court order, due to data privacy, reliability, and misinterpretation concerns. [ 71 ]


Three to five percent of the funding available for the  Human Genome Project  was set aside to study the many  social, ethical, and legal implications  that will result from the better understanding of human heredity the rapid expansion of genetic risk assessment by genetic testing which would be facilitated by this project. [ 72 ]


The  American Academy of Pediatrics  (AAP) and the  American College of Medical Genetics  (ACMG) have provided new guidelines for the ethical issue of  pediatric  genetic testing and  screening  of children in the United States. [ 73 ] [ 74 ]   Their guidelines state that performing pediatric genetic testing should be in the best interest of the child. AAP and ACMG recommend holding off on genetic testing for late-onset conditions until adulthood, unless diagnosing genetic disorders during childhood can reduce morbidity or mortality (e.g., to start early intervention). Testing asymptomatic children who are at risk of childhood onset conditions can also be warranted.
Both AAP and ACMG discourage the use of  direct-to-consumer  and home kit genetic tests because of concerns regarding the accuracy, interpretation and oversight of test content.
Guidelines also state that parents or guardians should be encouraged to inform their child of the results from the genetic test if the minor is of appropriate age. For ethical and legal reasons, health care providers should be cautious in providing minors with predictive genetic testing without the involvement of parents or guardians. Within the guidelines set by AAP and ACMG, health care providers have an obligation to inform parents or guardians on the implication of test results.
AAP and ACMG state that any type of predictive genetic testing should be offered with  genetic counseling  by  clinical genetics ,  genetic counselors  or health care providers. [ 74 ]


In Israel, DNA testing is used to determine if people are eligible for immigration. The policy where "many Jews from the former Soviet Union (FSU) are asked to provide DNA confirmation of their Jewish heritage in the form of paternity tests in order to immigrate as Jews and become citizens under Israel's  Law of Return " has generated controversy. [ 75 ] [ 76 ] [ 77 ] [ 78 ]


From the date that a sample is taken, results may take weeks to months, depending upon the complexity and extent of the tests being performed. Results for prenatal testing are usually available more quickly because time is an important consideration in making decisions about a pregnancy. Prior to the testing, the doctor or genetic counselor who is requesting a particular test can provide specific information about the cost and time frame associated with that test. [ 79 ]


This article incorporates  public domain material  from  What are the risks and limitations of genetic testing? .  United States Department of Health and Human Services .
  This article incorporates  public domain material  from  What is the cost of genetic testing, and how long does it take to get the results? .  United States Department of Health and Human Services .


Genetic use restriction technology  ( GURT ), also known as  terminator technology  or  suicide seeds , is designed to restrict access to "genetic materials and their associated  phenotypic traits ." [ 2 ]  The technology works by activating (or deactivating) specific genes using a controlled stimulus in order to cause second generation seeds to be either infertile or to not have one or more of the desired traits of the first generation plant. [ 3 ] [ 4 ]  GURTs can be used by agricultural firms to enhance protection of their innovations in  genetically modified organisms  by making it impossible for farmers to reproduce the desired traits on their own. [ 4 ]  Another possible use is to prevent the escape of genes from genetically modified organisms into the surrounding environment. [ 5 ]


The technology was originally developed under a cooperative research and development agreement between the  Agricultural Research Service  of the  United States Department of Agriculture  and  Delta & Pine Land Company  in the 1990s. The purpose of the development was to protect the intellectual property of biotechnology firms that the United States Department of Agriculture viewed as being a specifically American technological competence. [ 6 ]  The technology, while still being developed, is not yet commercially available due to the political and scientific controversies that accompanied its development. [ 7 ]


GURT was first reported on by the Subsidiary Body on Scientific, Technical and Technological Advice (SBSTTA) to the UN  Convention on Biological Diversity [ 3 ]  and discussed during the 8th Conference of the Parties to the United Nations  Convention on Biological Diversity  in  Curitiba , Brazil, March 20–31, 2006.


The GURT process is typically composed of four genetic components: a target gene, a promoter, a trait switch, and a genetic switch, sometimes with slightly different names given in different papers. [ 5 ]  A typical GURT involves the engineering of a plant that has a  target  gene  in its  DNA  that expresses when activated by a  promoter gene . However, it is separated from the target gene by a  blocker sequence  that prevents the promoter from accessing the target. When the plant receives a given external input, a  genetic switch  in the plant takes the input, amplifies it, and converts it into a biological signal. When a  trait switch  receives the amplified signal, it creates an  enzyme  that cuts the blocker sequence out. With the blocker sequence eliminated, the promoter gene allows the target gene to express itself in the plant. [ 5 ] [ 8 ]


In other versions of the process, an operator must bind to the trait switch in order for it to make the enzymes that cut out the blocker sequence. However, there are repressors that bind to the trait switch and prevent it from doing so. In this case, when the external input is applied, the repressors bond to the input instead of to the trait switch, allowing the enzymes to be created that cut the blocker sequence, thereby allowing the trait to be expressed. [ 6 ]


Other GURTs embody alternative approaches, such as letting the genetic switch directly affect the blocker sequence and bypass the need for a trait switch. [ 6 ]


There are two broad categories of GURTs: Variety-specific genetic use restriction technologies (V-GURTs) and Trait specific genetic use restriction technologies (T-GURTs). [ 9 ] [ 10 ]  The two variants have been described as follows [ 5 ] :


V-GURTs are designed to restrict the use of all genetic materials contained in an entire plant variety. Prior to being sold to growers, the seeds of V-GURTs are activated by the seed company. The seeds can germinate, and the plants grow and reproduce normally, but their offspring will be sterile... . Thus, farmers could not save seed from year-to-year to replant. In contrast, T-GURTs only restrict the use of particular traits conferred by a transgene, but seeds are fertile. Growers could replant seed from the previous harvest, but they would not contain the transgenic trait.


Variety-specific genetic use restriction technologies destroy seed development and plant fertility by means of a "genetic process triggered by a chemical inducer that will allow the plant to grow and to form seeds, but will cause the embryo of each of those seeds to produce a cell toxin that will prevent its germination if replanted, thus causing second generation seeds to be sterile... ." [ 6 ]  The toxin degrades the DNA or RNA of the plant. Thus, the seed from the crop is not viable and cannot be used as seeds to produce subsequent crops, but only for sale as food or fodder. [ 7 ] [ 11 ]


Trait specific genetic use restriction technologies modify a crop in such a way that the genetic enhancement engineered into the crop does not function until the plant is treated with a specific chemical. [ 7 ] [ 12 ]  The chemical acts as the external input, activating the target gene. One difference in T-GURTs is the possibility that the gene could be toggled on and off with different chemical inputs, resulting in the same toggling on or off an associated trait. With T-GURTs, seeds could possibly be saved for planting with a condition that the new plants do not get any enhanced traits unless the external input is added.


GURTs have a number of potential uses, though they have not yet been used in commercial agricultural products available on the market or in pharmaceutical applications. [ 13 ]  These uses include protection of intellectual property for biotechnological innovations, and bio-confinement (preventing escape of genetically engineered genes into nature).


The original aim of the developers of GURTs was the protection of intellectual property in agricultural biotechnology. That is, the developers sought to prevent farmers from reusing patented seeds in cases where  patents  for biological innovations did not exist or could not be easily enforced. [ 7 ]  This problem is not generally posed for farmers using  hybrid seeds  (which, in any case, are not fertile or do not breed true) and, thus, could not be used to grow subsequent crops. However, the V-GURTS make it impossible for farmers to use seeds they have produced to grow crops in subsequent seasons because the entire genome of the targeted cells is destroyed. The T-GURTs could be used by seed companies to allow for the commercialisation of seeds that are fertile, but that develop into plants with desired traits only when sprayed with an activator chemical sold by the company. [ 12 ]


An ongoing fear raised by GURTs and other biotechnologies is that the genes of genetically modified plants might escape into nature via  sexual reproduction  with compatible wild plants or with other cultivated plants. This is known as ' transgene  escape' and is among the highest priority risks posed by genetic engineering of plants. [ 4 ]  This risk of escape is one of the reasons that the GURT process has not yet been used in commercial applications (indeed, the main producing companies have vowed to not commercialise these products, though they still have related research programs). Ironically, GURTs – themselves a process for the genetic modification of plants – may also be used to secure the 'bio-confinement' of the transgenes of genetically modified plants. GURTs, because they control plant fertility in various ways, could be used to prevent the escape of transgenes into wild relatives and help reduce risks of deleterious impacts on  biodiversity . For bio-confinement, both "V- and T GURTs could be targeted to reproductive tissues, most typically pollen and seed (or embryo)." [ 5 ]  Crops modified to produce non-food products (eg. in pharmacology, therapeutic proteins, monoclonal antibodies and vaccines) could be armed with GURTs to prevent accidental transmission of these traits into crops meant for foods. [ 7 ]


Another possible advantage is that non-viable seeds produced on V-GURT plants may reduce the propagation of  volunteer plants . Volunteer plants can become an economic problem for larger-scale mechanized farming systems that incorporate  crop rotation . [ 7 ]  Furthermore, under warm, wet  harvest  conditions non V-GURT grain can sprout, lowering the quality of grain produced. It is likely that this problem would not occur with the use of V-GURT grain varieties. [ 7 ]


Another proposed use is in synthetic biology, where a restricted activator chemical must be added to the fermentation medium to produce a desired output chemical. [ 14 ]


As of 2006, GURT seeds have not been commercialized anywhere in the world due to opposition from farmers, consumers,  indigenous peoples ,  NGOs , and some governments. [ 6 ]  Using the technology, companies that manufacture genetic use restriction technologies could potentially acquire an advantageous position vis-a-vis farmers because the seeds sold could not be resown. V-GURTs would not have an immediate impact on the many farmers who use hybrid seeds, as they do not produce their own planting seeds, buying instead specialized hybrid seeds from seed production companies. However, approximately 80 percent of farmers in Brazil and Pakistan grow crops using seeds saved from previous harvests. [ 11 ]   Another concern is that farmers purchasing the seeds would be greatly impacted, given they would have to buy new seeds every year. It has been argued that this would result in higher prices in food. [ 15 ]


Some analysts have expressed concerns that GURT seeds might adversely impact biodiversity and threaten native species of plants. [ 16 ] [ 17 ]  However, proponents of the technology dispute these claims, arguing that because non-GMO hybrid plants are used in the same way and GURT seeds could help farmers deal with cross pollination, the benefits outweigh the potential negatives. [ 18 ]


In 2000, the  United Nations Convention on Biological Diversity  recommended a  de facto   moratorium  on field-testing and commercial sale of terminator seeds; the moratorium was re-affirmed and the language strengthened in March 2006, at the COP8 meeting of the UNCBD. [ 19 ]  Specifically, the moratorium recommended that, due to a lack of research on the technology's potential risks, no field testing of GURTs nor products using them should be allowed until there was a sufficiently justified reason to do so.  India  and  Brazil  have passed national laws to prohibit the technology. [ 11 ]


Genotyping  is the process of determining differences in the genetic make-up ( genotype ) of an individual by examining the individual's  DNA  sequence using  biological assays  and comparing it to another individual's sequence or a reference sequence. It reveals the  alleles  an individual has inherited from their parents. [ 1 ]  Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.


Current methods of genotyping include   restriction fragment length polymorphism  identification (RFLPI) of genomic DNA,  random amplified polymorphic detection  (RAPD) of genomic DNA,  amplified fragment length polymorphism  detection (AFLPD),  polymerase chain reaction  (PCR),  DNA sequencing ,  allele specific oligonucleotide  (ASO) probes, and  hybridization  to  DNA microarrays  or beads.  Genotyping is important in research  of genes and gene variants associated with disease.  Due to current technological limitations, almost all genotyping is partial.  That is, only a small fraction of an individual's genotype is determined, such as with (epi)GBS ( Genotyping by sequencing ) or  RADseq .  New  [ 2 ]   mass-sequencing technologies  promise to provide whole-genome genotyping (or  whole genome sequencing ) in the future.


Genotyping applies to a broad range of individuals, including microorganisms. For example,  viruses  and  bacteria  can be genotyped.  Genotyping in this context may help in controlling the spreading of pathogens, by tracing the origin of outbreaks. This area is often referred to as  molecular epidemiology  or  forensic   microbiology .


Humans can also be genotyped.  For example, when testing fatherhood or motherhood, scientists typically only need to examine 10 or 20 genomic regions (like  single-nucleotide polymorphism  (SNPs)), which represent a tiny fraction of the human  genome .


When genotyping  transgenic  organisms, a single genomic region may be all that needs to be examined to determine the genotype.  A single  PCR  assay is typically enough to genotype a transgenic  mouse ; the mouse is the  mammalian  model of choice for much of medical research today.


The ethical concerns of genotyping humans have been a topic of discussion. The rise of genotyping technologies will make it possible to  screen  large populations of people for  genetic diseases  and predispositions for disease. [ 3 ]  The benefits of population wide genotyping have been contended by ethical concerns on consent and general benefit of wide span screening. [ 3 ]  Genotyping identifies  mutations  that increase susceptibility of a person to develop a disease, but disease development is not guaranteed in most cases, which can cause psychological damage. [ 4 ]   Discrimination  can arise from various genetic markers identified by genotyping, such as athletic advantages or disadvantages in professional sports or risk of disease development later in life. [ 5 ] [ 4 ]  Much of the ethical concerns surrounding genotyping arise from information availability, as in who can access the  genotype  of an individual in various contexts. [ 4 ]


Genotyping is used in the medical field to identify and control the spread of  tuberculosis  (TB). Originally, genotyping was only used to confirm outbreaks of tuberculosis; but with the evolution of genotyping technology it is now able to do far more. Advances in genotyping technology led to the realization that many cases of tuberculosis, including infected individuals living in the same household, were not actually linked. [ 6 ]  This caused the formation of universal genotyping in an attempt to understand transmission dynamics. Universal genotyping revealed complex transmission dynamics based on things like socio-epidemiological factors. This led to the use of polymerase chain reactions (PCR) which allowed for faster detection of tuberculosis. This rapid detection method is used to prevent TB. [ 6 ]  The addition of whole genome sequencing (WGS) allowed for identification of strains of TB which could then be put in a chronological cluster map. These cluster maps show the origin of cases and the time in which those cases arose. This gives a much clearer picture of transmission dynamics and allows for better control and prevention of transmission. All of these different forms of genotyping are used together to detect TB, prevent its spread and trace the origin of infections. This has helped to reduce the number of TB cases. [ 6 ]


Many types of genotyping are used in  agriculture . One type that is used is genotyping by sequencing because it aids agriculture with crop breeding. For this purpose, single nucleotide polymorphisms (SNPs) are used as markers and  RNA sequencing  is used to look at gene expression in crops. [ 7 ]  The knowledge gained from this type of genotyping allows for selective breeding of crops in ways which benefit agriculture. In the case of alfalfa, the cell wall was improved through selective breeding that was made possible by this type of genotyping. [ 7 ]  These techniques have also resulted in the discovery of genes that provide resistance to diseases. A gene called Yr15 was discovered in wheat, which protects against a disease called yellow wheat rust. Selective breeding for the Yr15 gene then prevented yellow wheat rust, benefiting agriculture. [ 7 ]


Golgi's method  is a  silver staining  technique that is used to visualize  nervous tissue  under light microscopy. The method was discovered by  Camillo Golgi , an  Italian   physician  and  scientist , who published the first picture made with the technique in 1873. [ 1 ]   It was initially named the  black reaction  ( la reazione nera ) by Golgi, but it became better known as the  Golgi stain  or later, Golgi method.


Golgi staining was used by  Spanish   neuroanatomist   Santiago Ramón y Cajal  (1852–1934) to discover a number of novel facts about the organization of the nervous system, inspiring the birth of the  neuron doctrine . Ultimately, Ramón y Cajal improved the technique by using a method he termed "double impregnation". Ramón y Cajal's staining technique, still in use, is called Cajal's Stain. [ citation needed ]


The cells in nervous tissue are densely packed and little information on their structures and interconnections can be obtained if all the cells are stained. Furthermore, the thin filamentary extensions of neural cells, including the  axon  and the  dendrites  of neurons, are too slender and transparent to be seen with normal staining techniques. Golgi's method stains a limited number of cells at random in their entirety. The mechanism by which this happens is still largely unknown. [ 2 ]  Dendrites, as well as the cell soma, are clearly stained in brown and black and can be followed in their entire length, which allowed neuroanatomists to track connections between neurons and to make visible the complex networking structure of many parts of the  brain  and  spinal cord .


Golgi's staining is achieved by impregnating aldehyde fixed nervous tissue with  potassium dichromate  and  silver nitrate . Cells thus stained are filled by  microcrystallization  of  silver chromate .


According to SynapseWeb, [ 3 ]  this is the recipe for Golgi's staining technique:


This technique has since been refined to substitute the silver precipitate with gold by immersing the sample in  gold chloride  then  oxalic acid , followed by removal of the silver by  sodium thiosulphate . This preserves a greater degree of fine structure with the ultrastructural details marked by small particles of gold.  [ 4 ]


Ramón y Cajal said of the Golgi method:


In  genetics ,  haplotype estimation  (also known as "phasing") refers to the process of statistical estimation of  haplotypes  from  genotype  data. The most common situation arises when genotypes are collected at a set of polymorphic sites from a group of individuals. For example in human genetics,  genome-wide association studies  collect genotypes in thousands of individuals at between 200,000-5,000,000 SNPs using microarrays. Haplotype estimation methods are used in the analysis of these datasets and allow  genotype imputation [ 1 ] [ 2 ]  of alleles from reference databases such as the  HapMap Project  and  the 1000 Genomes Project .


Genotypes measure the unordered combination of alleles at each locus, whereas haplotypes represent the genetic information on multiple loci that have been inherited together from an individual's parents. Theoretically the number of possible haplotypes equals to the product of allele numbers of each locus in consideration. Specially, most of the SNPs are bi-allelic; Therefore when considering

 N

 {\displaystyle N}

   heterozygous  bi-allelic loci, there will be

 2

 N

 {\displaystyle 2^{N}}

  possible pairs of haplotypes that could underlie the genotypes. For example, when considering two bi-allelic loci A and B (

 N
 =
 2

 {\displaystyle N=2}

 ), of which the genotypes are  a 1  and  a 2 ,  b 1  and  b 2 ,  respectively, we will have the following haplotypes:  a 1 _b 1 , a 1 _b 2 , a 2 _b 1 ,  and  a 2 _b 2  ( "_"  indicates that the alleles are on the same chromosome).


Many statistical methods have been proposed for estimation of haplotypes. Some of the earliest approaches used a simple multinomial model in which each possible haplotype consistent with the sample was given an unknown frequency parameter and these parameters were estimated with an  Expectation–maximization algorithm . These approaches were only able to handle small numbers of sites at once, although sequential versions were later developed, specifically the SNPHAP method.


The most accurate and widely used methods for haplotype estimation utilize some form of  hidden Markov model  (HMM) to carry out inference. For a long time PHASE [ 3 ]  was the most accurate method. PHASE was the first method to utilize ideas from  coalescent theory  concerning the joint distribution of haplotypes. This method used a  Gibbs sampling  approach in which each individuals haplotypes were updated conditional upon the current estimates of haplotypes from all other samples. Approximations to the distribution of a haplotype conditional upon a set of other haplotypes were used for the conditional distributions of the Gibbs sampler. PHASE was used to estimate the haplotypes from the  HapMap Project . PHASE was limited by its speed and was not applicable to datasets from genome-wide association studies.


The fastPHASE  [ 4 ]  and BEAGLE methods  [ 5 ]  introduced haplotype cluster models applicable to  GWAS -sized datasets. Subsequently the IMPUTE2 [ 6 ]  and MaCH [ 7 ]  methods were introduced that were similar to the PHASE approach but much faster. These methods iteratively update the haplotype estimates of each sample conditional upon a subset of K haplotype estimates of other samples. IMPUTE2 introduced the idea of carefully choosing which subset of haplotypes to condition on to improve accuracy. Accuracy increases with K but with quadratic

 O
 (

 K

 2

 )

 {\displaystyle O(K^{2})}

  computational complexity.


The SHAPEIT1 method made a major advance by introducing a linear

 O
 (
 K
 )

 {\displaystyle O(K)}

  complexity method that operates only on the space of haplotypes consistent with an individual’s genotypes. [ 8 ]  The HAPI-UR method subsequently proposed a very similar method. [ 9 ]  SHAPEIT2  [ 10 ]  combines the best features of SHAPEIT1  and IMPUTE2 to improve efficiency and accuracy.


Helicase-dependent amplification  (HDA) is a method for  in vitro   DNA  amplification (like the  polymerase chain reaction ) that takes place at a constant temperature.


The polymerase chain reaction is the most widely used method for  in vitro  DNA amplification for purposes of molecular biology and biomedical research. [ 1 ]   This process involves the separation of the double-stranded DNA in high heat into single strands (the denaturation step, typically achieved at 95–97 °C), annealing of the primers to the single stranded DNA (the annealing step) and copying the single strands to create new double-stranded  DNA (the extension step that requires the DNA polymerase) requires the reaction to be done in a  thermal cycler .  These bench-top machines are large, expensive and costly to run and maintain, limiting the potential applications of DNA amplification in situations outside the laboratory (e.g., in the identification of potentially hazardous micro-organisms at the scene of investigation, or at the point of care of a patient). Although PCR is usually associated with thermal cycling, the original patent by Mullis et al.  [ 2 ]  disclosed the use of a helicase as a means for denaturation of double stranded DNA thereby including isothermal nucleic acid amplification.
 In vivo , DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate the DNA by unwinding the DNA double helix. [ 3 ]    HDA was developed from this concept, using a  helicase  (an  enzyme ) to denature the DNA.


Strands of double-stranded DNA are first separated by a DNA helicase and coated by single-stranded DNA (ssDNA)-binding proteins. In the second step, two sequence-specific primers hybridise to each border of the DNA template. DNA polymerases are then used to extend the primers annealed to the templates to produce a double-stranded DNA and the two newly synthesized DNA products are then used as substrates by DNA helicases, entering the next round of the reaction. Thus, a simultaneous chain reaction develops, resulting in exponential amplification of the selected target sequence (see Vincent  et al. ., 2004 [ 4 ]  for a schematic diagram).


Since the publication of its discovery, HDA technology has been used for a "simple, easy to adapt nucleic acid test for the detection of  Clostridioides difficile ". [ 5 ]   Other applications include the rapid detection of  Staphylococcus aureus   by the amplification and detection of a short DNA sequence specific to the bacterium. [ 6 ]  The advantages of HDA is that it provides a rapid method of nucleic acid amplification of a specific target at an isothermic temperature that does not require a thermal cycler.  However, the optimisation of primers and sometimes buffers is required beforehand by the researcher.  Normally primer and buffer optimisation is tested and achieved through PCR, raising the question of the need to spend extra on a separate system to do the actual amplification.  Despite the selling point that HDA negates the use of a thermal cycler and therefore allows research to be conducted in the field, much of the work required to detect potentially hazardous microorganisms is carried out in a research/hospital lab setting regardless. At present, mass diagnoses from a great number of samples cannot yet be achieved by HDA, whereas PCR reactions carried out in  thermal cycler  that can hold multi-well sample plates allows for the amplification and detection of the intended DNA target from a maximum of 96 samples.  The cost of purchasing reagents for HDA are also relatively expensive to that of PCR reagents, more so since it comes as a ready-made kit.


Hin recombinase  is a 21kD  protein  composed of 198  amino acids  that is found in the bacteria  Salmonella . Hin belongs to the  serine recombinase  family (B2) of  DNA invertases  in which it relies on the  active site   serine  to initiate DNA cleavage and recombination. The related protein,  gamma-delta resolvase  shares high similarity to Hin, of which much structural work has been done, including structures bound to DNA and  reaction intermediates . Hin functions to invert a 900 base pair ( bp ) DNA segment within the salmonella  genome  that contains a  promoter  for  downstream   flagellar  genes, fljA and fljB. Inversion of the intervening DNA alternates the direction of the promoter and thereby alternates  expression  of the flagellar genes. This is advantageous to the bacterium as a means of escape from the  host   immune response .


Hin functions by binding to two 26bp imperfect inverted repeat sequences as a  homodimer . These hin binding sites flank the invertible segment which not only encodes the Hin gene itself, but also contains an enhancer element to which the bacterial  Fis  proteins binds with nanomolar affinity. Four molecules of Fis bind to this site as a homodimers and are required for the recombination reaction to proceed.


The initial reaction requires binding of Hin and Fis to their respective DNA sequences and assemble into a higher-order  nucleoprotein  complex with branched plectonemic  supercoils  with the aid of the DNA bending protein HU. At this point, it is believed that the Fis protein modulates subtle contacts to activate the reaction, possibly through direct interactions with the Hin protein. Activation of the 4 catalytic serine residues within the Hin  tetramer  make a 2-bp double stranded DNA break and forms a covalent reaction intermediate. The DNA cleavage event also requires the divalent metal  cation   magnesium . A large  conformational change  reveals a large  hydrophobic  interface that allows for subunit rotation which may be driven by superhelical torsion within the protein-DNA complex. After this 180° rotation, Hin returns to its native conformation and re-ligates the cleaved DNA, without the aid of high energy cofactors and without the loss of any DNA.


In  molecular biology , a  hybridization probe  ( HP ) is a fragment of  DNA  or  RNA , usually 15–10000  nucleotides  long, which can be  radioactively  or  fluorescently labeled . HPs can be used to detect the presence of   nucleotide  sequences in analyzed RNA or DNA that are  complementary  to the sequence in the probe. [ 1 ]  The labeled probe is first  denatured  (by heating or under  alkaline  conditions such as exposure to  sodium hydroxide ) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA ( Southern blotting ) or RNA ( northern blotting ) immobilized on a membrane or  in situ .


To detect  hybridization  of the probe to its target sequence, the probe is tagged (or "labeled") with a  molecular marker  of either radioactive or (more recently) fluorescent molecules. Commonly used markers are  32 P  (a  radioactive   isotope  of  phosphorus  incorporated into the  phosphodiester  bond in the probe DNA),  digoxigenin ,  a non-radioactive,  antibody -based marker, biotin or fluorescein. DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via  autoradiography  or other imaging techniques. Normally, either X-ray pictures are taken of the filter, or the filter is placed under UV light. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied—high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between  nucleic acid  sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar.


Hybridization probes used in  DNA microarrays  refer to DNA covalently attached to an inert surface, such as coated  glass slides  or  gene chips , to which a mobile  cDNA  target is hybridized. Depending on the  method , the probe may be  synthesized  using the  phosphoramidite  method, or it can be generated and labeled by  PCR  amplification or  cloning  (both are older methods). In order to increase the  in vivo  stability of the probe RNA is not used. Instead,  RNA analogues  may be used, in particular  morpholino - derivatives. Molecular DNA- or RNA-based probes are routinely used in screening gene libraries, detecting nucleotide sequences with  blotting methods , and in other gene technologies, such as nucleic acid and tissue  microarrays .


Within the field of  microbial ecology , oligonucleotide probes are used in order to determine the presence of microbial species, genera, or microorganisms classified on a more broad level, such as  bacteria ,  archaea , and  eukaryotes  via  fluorescence in situ hybridization  (FISH). [ 2 ]  rRNA probes have enabled scientists to visualize microorganisms, yet to be cultured in laboratory settings, by retrieval of rRNA sequences directly from the environment. [ 3 ]  Examples of these types of microorganisms include:


In some instances, differentiation between species may be problematic when using  16S rRNA  sequences due to similarity. In such instances,  23S rRNA  may be a better alternative. [ 6 ]  The global standard library of rRNA sequences is constantly becoming larger and continuously being updated, and thus the possibility of a random hybridization event between a specifically-designed probe (based on complete and current data from a range of test organisms) and an undesired/unknown target organism cannot be easily dismissed. [ 7 ]  On the contrary, it is plausible that there exist microorganisms, yet to be identified, which are phylogenetically members of a probe target group, but have partial or near-perfect target sites, usually applies when designing group-specific probes.


Probably the greatest practical limitation to this technique is the lack of available automation. [ 8 ]


In forensic science, hybridization probes are used, for example, for detection of short tandem repeats ( microsatellite ) regions [ 9 ]  and in restriction fragment length polymorphism ( RFLP ) methods, all of which are widely used as part of  DNA profiling  analysis.


iCLIP [ 1 ] [ 2 ] [ 3 ]  (individual-nucleotide resolution crossLinking and immunoprecipitation) is a variant of the original CLIP method used for identifying protein-RNA interactions, [ 4 ]  which uses  UV light  to covalently bind proteins and RNA molecules  to identify RNA binding sites of proteins. This  crosslinking  step has generally less background than standard  RNA immunoprecipitation  (RIP) protocols, because the covalent bond formed by UV light allows RNA to be fragmented, followed by stringent purification, and this also enables CLIP to identify the positions of protein-RNA interactions. [ 5 ]  As with all  CLIP  methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes by stringent washing during immunoprecipitation followed by  SDS-PAGE  and transfer to nitrocellulose. The labelled protein-RNA complexes are then visualised for quality control, excised from nitrocellulose, and treated with proteinase to release the RNA, leaving only a few amino acids at the crosslink site of the RNA. [ 6 ]


The RNA is then reverse transcribed, causing most cDNAs to truncate at the crosslink site, and the key innovation and unique feature in the development of iCLIP was to enable such truncated cDNAs to be PCR amplified and sequenced using a next-generation sequencing platform. iCLIP also added a random sequence (unique molecular identifier,  UMI ) along with experimental barcodes to the primer used for reverse transcription, thereby barcoding unique cDNAs to minimise any errors or quantitative biases of PCR, and thus improving the quantification of binding events. Enabling amplification of truncated cDNAs led to identification of the sites of RNA-protein interactions at high resolution by analysing the starting position of truncated cDNAs, as well as their precise quantification using UMIs with software called " iCount ". [ 1 ]  All these innovations of iCLIP were adopted by later variants of CLIP [ 6 ]  such as eCLIP [ 7 ]  and irCLIP. [ 8 ]  An additional approach to identify protein-RNA crosslink sites is the mutational analysis of read-through cDNAs, such as nucleotide transitions in  PAR-CLIP , [ 9 ]  or other types of errors that can be introduced by reverse transcriptase when it reads through the crosslink site in standard  HITS-CLIP  method with the Crosslink induced mutation site (CIMS) analysis. [ 10 ]


The quantitative nature of iCLIP enabled pioneering comparison across samples at the level of full RNAs, [ 11 ]  or to study competitive binding of multiple RNA-binding proteins [ 12 ]  or subtle changes in binding of a mutant protein at the level of binding peaks. [ 13 ]  An improved variant of iCLIP (iiCLIP) was recently developed to improve the efficiency and convenience of cDNA library preparation, for example by enzymatically removing adaptor after ligation to minimise artefacts caused by adaptor carry-over, introducing the non-radioactive visualisation of the protein-RNA complex (as done originally by irCLIP [ 8 ] ), increasing efficiency of ligation, proteinase and reverse transcription reactions, and enabling bead-based purification of cDNAs. [ 14 ]


Analysis of CLIP sequencing data benefits from use of customised computational software, much of which is available as part of the  Nextflow pipeline for CLIP analysis , and specialised software is available for  rapid demultiplexing  of complex multiplexed libraries, [ 15 ]  comparative visualisation of  crosslinking profiles  across RNAs, [ 16 ]   identification of the peaks  of clustered protein-RNA crosslink sites, and  identification of sequence motifs  enriched around prominent crosslinks. [ 17 ]  Moreover,  iMaps  provides a free CLIP analysis web platform and well-curated community database to facilitate studies of RNA regulatory networks across organisms, with a backend based on the Nextflow pipeline. It is applicable to the many variant protocols of CLIP (such as iCLIP, eCLIP, etc), and can be used to analyse unpublished data in a secure manner, or to obtain public CLIP data in a well-annotated format, along with various forms of quality control, visualisation and comparison. Questions on the experimental and computational challenges are collated on the Q&A  CLIP Forum .


In situ  hybridization (ISH)  is a type of  hybridization  that uses a labeled  complementary DNA ,  RNA  or modified nucleic acid strand (i.e., a  probe ) to localize a specific DNA or RNA sequence in a portion or section of  tissue  ( in situ ) or if the tissue is small enough (e.g., plant seeds,  Drosophila  embryos), in the entire tissue (whole mount ISH), in cells, and in  circulating tumor cells  (CTCs). This is distinct from  immunohistochemistry , which usually localizes proteins in tissue sections.


In situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation, and function of genes. The key techniques currently in use include  in situ  hybridization to mRNA with  oligonucleotide  and RNA probes (both radio-labeled and hapten-labeled), analysis with light and electron microscopes, whole mount  in situ  hybridization, double detection of RNAs and RNA plus protein, and fluorescent  in situ  hybridization to detect chromosomal sequences. DNA ISH can be used to determine the  structure  of chromosomes.  Fluorescent DNA ISH  (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (RNA  in situ  hybridization) is used to measure and localize RNAs (mRNAs, lncRNAs, and miRNAs) within tissue sections, cells, whole mounts, and circulating tumor cells (CTCs).  In situ  hybridization was invented by American biologists  Mary-Lou Pardue  and  Joseph G. Gall . [ 1 ] [ 2 ] [ 3 ]


In situ hybridization is a powerful technique for identifying specific mRNA species within individual cells in tissue sections, providing insights into physiological processes and disease pathogenesis. However, in situ hybridization requires that many steps be taken with precise optimization for each tissue examined and for each probe used. In order to preserve the target mRNA within tissues, it is often required that crosslinking fixatives (such as  formaldehyde ) be used. [ 4 ]


In addition, in-situ hybridization on tissue sections require that tissue slices be very thin, usually 3 μm to 7 μm in thickness. Common methods of preparing tissue sections for in-situ hybridization processing include cutting specimens with a cryostat or a  Compresstome tissue slicer . A  cryostat  takes fresh or fixed tissue and immerses it into liquid nitrogen for flash freezing. Then tissue is embedded in freeze media called OCT and thin sections are cut. Obstacles include getting freeze artifacts on tissue that may interfere with proper mRNA staining. The  Compresstome  cuts tissue into thin slices without a freeze process; free-floating sections are cut after being embedded in agarose for stability. This method avoids freezing tissue and thus associated freeze artifacts. The process is permanent and irreversible once its complete. [ 5 ]


For hybridization  histochemistry , sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. As noted above, the probe is either a labeled  complementary DNA  or, now most commonly, a complementary  RNA   ( riboprobe ). The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe). Solution parameters such as temperature, salt, and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e., only exact sequence matches will remain bound). Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases (e.g.,  digoxigenin ) is localized and quantified in the tissue using either  autoradiography ,  fluorescence microscopy , or  immunohistochemistry , respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.


An alternative technology,  branched DNA assay , can be used for RNA (mRNA, lncRNA, and miRNA )  in situ  hybridization assays with single molecule sensitivity without the use of radioactivity. This approach (e.g., ViewRNA assays) can be used to visualize up to four targets in one assay, and it uses patented probe design and bDNA signal amplification to generate sensitive and specific signals. Samples (cells, tissues, and CTCs) are fixed, then treated to allow RNA target accessibility (RNA un-masking). Target-specific probes hybridize to each target RNA. Subsequent signal amplification is predicated on specific hybridization of adjacent probes (individual  oligonucleotides  [oligos] that bind side by side on RNA targets). A typical target-specific probe will contain 40 oligonucleotides, resulting in 20 oligo pairs that bind side-by-side on the target for detection of mRNA and lncRNA, and 2 oligos or a single pair for miRNA detection. Signal amplification is achieved via a series of sequential hybridization steps. A pre-amplifier molecule hybridizes to each oligo pair on the target-specific RNA, then multiple amplifier molecules hybridize to each pre-amplifier. Next, multiple label probe oligonucleotides (conjugated to alkaline phosphatase or directly to fluorophores) hybridize to each amplifier molecule. A fully assembled signal amplification structure “Tree” has 400 binding sites for the label probes. When all target-specific probes bind to the target mRNA transcript, an 8,000 fold signal amplification occurs for that one transcript. Separate but compatible signal amplification systems enable the multiplex assays.  The signal can be visualized using a fluorescence or brightfield microscope.


The protocol takes around 2–3 days and takes some time to set up. Some companies sell robots to automate the process (e.g.,  CEM  InsituPro). As a result, large-scale screenings have been conducted in laboratories on thousands of genes. The results can usually be accessed via websites (see external links). [ 6 ]


Iterons  are directly repeated  DNA sequences  which play an important role in regulation of  plasmid  copy number in  bacterial   cells . It is one among the three negative regulatory elements found in plasmids which control its copy number. The others include  antisense RNAs  and  ctRNAs . Iterons complex with cognate replication (Rep) initiator  proteins  to achieve the required regulatory effect. [ 1 ] [ 2 ]


Iterons have an important role in plasmid replication. An iteron-containing plasmid  origin of replication  can be found containing about five iterons about 20  base pairs  in length total. These iterons provide a saturation site for initiator receptor proteins and promote replication thus increasing plasmid copy number in a given cell. [ 1 ]


There are four main limiting factors leading to no initiation of replication in iterons: [ 1 ]


Transcriptional auto-repression is thought to reduce initiator synthesis by repressing the formation of the Rep proteins. Since these proteins work to promote binding of replication machinery, replication can be halted in this form. Another factor used to stop replication is known as dimerization. It works to dimerize these Rep proteins and as a result  monomers  of these proteins are no longer in a high enough concentration to initiate replication. [ 2 ]  Another limiting factor, titration, occurs after replication and works to prevent saturation by distributing monomers to daughter origins so that no are fully saturated. Finally, handcuffing refers to pairing origins leading to inactivation. This is mediated by monomers and inactivation is due to  steric hindrance  between the origins. [ 1 ] [ 2 ]


Another less prevalent limitation thought to be present in these iterons is the presence of extra repeats. If a plasmid contains an extra supply of iterons outside of the saturation site it has been shown this can decrease plasmid copy number. In contrast, removing these extra iterons will increase copy number. [ 1 ]


Plasmids are known to have very similar structure when under control of Iterons. This structure consists of an origin of replication upstream of a gene that codes for a replication initiator protein. The iterons themselves are known to cover about half of the origin of replication. [ 2 ]  Usually, iterons on the same plasmid are highly conserved, whereas comparing iterons on different plasmids still exhibit  homology  yet are not as highly conserved. This suggests that iterons could be  evolutionarily  related. [ 3 ]


The replication initiator protein (Rep) plays a key role in initiation of replication in plasmids. In its monomer form, Rep binds an iteron and promotes replication. The protein itself is known to contain two independent  N-terminal  and  C-terminal   globular domains  that subsequently bind to two domains of the iteron. The dimer version of the protein is generally inactive in iteron binding, however it is known to bind to the repE  operator . This operator contains half of the iteron sequence making it able to bind the dimer and promote gene expression. [ 2 ] [ 4 ]


Plasmids containing iterons are all organized very similarly in structure. [ 2 ]    The gene for Rep proteins is usually found directly downstream of the origin of replication. [ 5 ]  This means that the iterons themselves are known to regulate the synthesis of the rep proteins. [ 6 ] [ 7 ]


A  karyotype  is the general appearance of the complete set of  chromosomes  in the cells of a  species  or in an individual organism, mainly including their sizes, numbers, and shapes. [ 1 ] [ 2 ]   Karyotyping  is the process by which a karyotype is discerned by determining the chromosome complement of an individual, including the number of chromosomes and any abnormalities.


A  karyogram  or  idiogram  is a graphical depiction of a karyotype, wherein chromosomes are generally organized in pairs, ordered by size and position of centromere for chromosomes of the same size. Karyotyping generally combines  light microscopy  and  photography  in the  metaphase  of the  cell cycle , and results in a  photomicrographic  (or simply micrographic) karyogram. In contrast, a  schematic  karyogram is a designed graphic representation of a karyotype. In schematic karyograms, just one of the sister  chromatids  of each chromosome is generally shown for brevity, and in reality they are generally so close together that they look as one on photomicrographs as well unless the resolution is high enough to distinguish them. The study of whole sets of chromosomes is sometimes known as  karyology .


Karyotypes describe the  chromosome count of an organism  and what these chromosomes look like under a light  microscope . Attention is paid to their length, the position of the  centromeres , banding pattern, any differences between the  sex chromosomes , and any other physical characteristics. [ 3 ]  The preparation and study of karyotypes is part of  cytogenetics .


The basic number of chromosomes in the  somatic  cells of an individual or a species is called the  somatic number  and is designated  2n . In the  germ-line  (the sex cells) the chromosome number is  n  (humans: n = 23). [ 4 ] [ 5 ] p28  Thus, in humans 2n = 46.


So, in normal  diploid  organisms,  autosomal  chromosomes are present in two copies. There may, or may not, be  sex chromosomes .  Polyploid  cells have multiple copies of chromosomes and  haploid  cells have single copies.


Karyotypes can be used for many purposes; such as to study  chromosomal aberrations ,  cellular  function,  taxonomic  relationships,  medicine  and to gather information about past  evolutionary  events ( karyosystematics ). [ 6 ]


The study of karyotypes is made possible by  staining . Usually, a suitable  dye , such as  Giemsa , [ 8 ]  is applied after  cells  have been arrested during  cell division  by a solution of  colchicine  usually in  metaphase  or  prometaphase  when most condensed. In order for the  Giemsa  stain to adhere correctly, all chromosomal proteins must be digested and removed. For humans,   white blood cells  are used most frequently because they are easily induced to divide and grow in  tissue culture . [ 9 ]  Sometimes observations may be made on non-dividing ( interphase ) cells. The sex of an unborn  fetus  can be predicted by observation of interphase cells (see  amniotic centesis  and  Barr body ).


Six different characteristics of karyotypes are usually observed and compared: [ 10 ]


A full account of a karyotype may therefore include the number, type, shape and banding of the chromosomes, as well as other cytogenetic information.


Variation is often found:


Both the micrographic and schematic karyograms shown in this section have a standard chromosome layout, and display darker and lighter regions as seen on  G banding , which is the appearance of the chromosomes after treatment with  trypsin  (to partially digest the chromosomes) and  staining  with  Giemsa stain . Compared to darker regions, the lighter regions are generally more  transcriptionally  active, with a greater ratio of  coding DNA  versus  non-coding DNA , and a higher  GC content . [ 11 ]


Both the micrographic and schematic karyograms show the normal human  diploid  karyotype, which is the typical composition of the  genome  within a normal cell of the human body, and which contains 22 pairs of  autosomal  chromosomes and one pair of  sex chromosomes  (allosomes). A major exception to diploidy in humans is  gametes  (sperm and egg cells) which are haploid with 23 unpaired chromosomes, and this  ploidy  is not shown in these karyograms. The micrographic karyogram is converted into  grayscale , whereas the schematic karyogram shows the purple hue as typically seen on Giemsa stain (and is a result of its azure B component, which stains DNA purple). [ 14 ]


The schematic karyogram in this section is a graphical representation of the idealized karyotype. For each chromosome pair, the scale to the left shows the length in terms of million  base pairs , and the scale to the right shows the designations of the  bands and sub-bands . Such bands and sub-bands are used by the  International System for Human Cytogenomic Nomenclature  to describe locations of  chromosome abnormalities . Each row of chromosomes is vertically aligned at  centromere  level.


Based on the karyogram characteristics of size, position of the  centromere  and sometimes the presence of a  chromosomal satellite  (a segment distal to a  secondary constriction ), the human chromosomes are classified into the following groups: [ 15 ]


Alternatively, the human genome can be classified as follows, based on pairing, sex differences, as well as location within the  cell nucleus  versus inside  mitochondria :


Schematic karyograms generally display a DNA copy number corresponding to the  G 0  phase  of the cellular state (outside of the replicative  cell cycle ) which is the most common state of cells. The schematic karyogram in this section also shows this state. In this state (as well as during the G 1  phase of the  cell cycle ), each cell has 2 autosomal chromosomes of each kind (designated 2n), where each chromosome has one copy of each  locus , making a total copy number of 2 for each locus (2c). At top center in the schematic karyogram, it also shows the chromosome 3 pair after having undergone  DNA synthesis , occurring in the  S phase  (annotated as S) of the cell cycle. This interval includes the  G 2  phase  and  metaphase  (annotated as "Meta."). During this interval, there is still 2n, but each chromosome will have 2 copies of each locus, wherein each  sister chromatid  (chromosome arm) is connected at the centromere, for a total of 4c. [ 17 ]  The chromosomes on micrographic karyograms are in this state as well, because they are generally micrographed in metaphase, but during this phase the two copies of each chromosome are so close to each other that they appear as one unless the image resolution is high enough to distinguish them. In reality, during the G 0  and G 1  phases, nuclear DNA is dispersed as  chromatin  and does not show visually distinguishable chromosomes even on micrography.


The copy number of the  human mitochondrial genome  per human cell varies from 0 (erythrocytes) [ 18 ]  up to 1,500,000 ( oocytes ), mainly depending on the number of mitochondria per cell. [ 19 ]


Although the  replication  and  transcription  of  DNA  is highly standardized in  eukaryotes , the same cannot be said for their karyotypes, which are highly variable. There is variation between species in chromosome number, and in detailed organization, despite their construction from the same  macromolecules . This variation provides the basis for a range of studies in evolutionary  cytology .


In some cases there is even significant variation within species. In a review, Godfrey and Masters conclude:


In our view, it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed ... But, used in conjunction with other phylogenetic data, karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species, which were previously inexplicable. [ 20 ]


Although much is known about karyotypes at the descriptive level, and it is clear that changes in karyotype organization has had effects on the evolutionary course of many species, it is quite unclear what the general significance might be.


We have a very poor understanding of the causes of karyotype evolution, despite many careful investigations ... the general significance of karyotype evolution is obscure.


Instead of the usual gene repression, some organisms go in for large-scale elimination of  heterochromatin , or other kinds of visible adjustment to the karyotype.


A spectacular example of variability between closely related species is the  muntjac , which was investigated by  Kurt Benirschke  and  Doris Wurster . The diploid number of the Chinese muntjac,  Muntiacus reevesi , was found to be 46, all  telocentric . When they looked at the karyotype of the closely related Indian muntjac,  Muntiacus muntjak , they were astonished to find it had female = 6, male = 7 chromosomes. [ 28 ]


They simply could not believe what they saw ... They kept quiet for two or three years because they thought something was wrong with their tissue culture ... But when they obtained a couple more specimens they confirmed [their findings].


The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. The low record is held by the  nematode   Parascaris univalens , where the  haploid  n = 1; and an ant:  Myrmecia pilosula . [ 30 ]  The high record would be somewhere amongst the  ferns , with the adder's tongue fern  Ophioglossum  ahead with an average of 1262 chromosomes. [ 31 ]  Top score for animals might be the  shortnose sturgeon   Acipenser brevirostrum  at 372 chromosomes. [ 32 ]  The existence of supernumerary or  B chromosomes  means that chromosome number can vary even within one interbreeding population; and  aneuploids  are another example, though in this case they would not be regarded as normal members of the population.


The fundamental number,  FN , of a karyotype is the number of visible major chromosomal arms per set of chromosomes. [ 33 ] [ 34 ]  Thus, FN ≤ 2 × 2n, the difference depending on the number of chromosomes considered single-armed ( acrocentric  or  telocentric ) present. Humans have FN = 82, [ 35 ]  due to the presence of five acrocentric chromosome pairs:  13 ,  14 ,  15 ,  21 , and  22  (the human  Y chromosome  is also acrocentric). The fundamental autosomal number or autosomal fundamental number,  FNa [ 36 ]  or  AN , [ 37 ]  of a karyotype is the number of visible major chromosomal arms per set of  autosomes  (non- sex-linked chromosomes ).


Ploidy  is the number of complete sets of chromosomes in a cell.


Polyploid series in related species which consist entirely of multiples of a single basic number are known as  euploid .


In many instances, endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). The cells do not always contain exact multiples (powers of two), which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate.


This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. [ 46 ]


The phenomenon occurs sporadically throughout the  eukaryote  kingdom from  protozoa  to humans; it is diverse and complex, and serves  differentiation  and  morphogenesis  in many ways. [ 47 ]


Aneuploidy  is the condition in which the chromosome number in the cells is not the typical number for the species. This would give rise to a  chromosome abnormality  such as an extra chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually cause a defect in development.  Down syndrome  and  Turner syndrome  are examples of this.


Aneuploidy may also occur within a group of closely related species. Classic examples in plants are the genus  Crepis , where the gametic (= haploid) numbers form the series x = 3, 4, 5, 6, and 7; and  Crocus , where every number from x = 3 to x = 15 is represented by at least one species. Evidence of various kinds shows that trends of evolution have gone in different directions in different groups. [ 48 ]  In primates, the  great apes  have 24x2 chromosomes whereas humans have 23x2.  Human chromosome 2  was formed by a merger of ancestral chromosomes, reducing the number. [ 49 ]


Some species are  polymorphic  for different chromosome structural forms. [ 50 ]  The structural variation may be associated with different numbers of chromosomes in different individuals, which occurs in the ladybird beetle  Chilocorus stigma , some  mantids  of the genus  Ameles , [ 51 ]  the European shrew  Sorex araneus . [ 52 ]  There is some evidence from the case of the  mollusc   Thais lapillus  (the  dog whelk ) on the  Brittany  coast, that the two chromosome morphs are  adapted  to different habitats. [ 53 ]


The detailed study of chromosome banding in insects with  polytene chromosomes  can reveal relationships between closely related species: the classic example is the study of chromosome banding in  Hawaiian drosophilids  by  Hampton L. Carson .


In about 6,500 sq mi (17,000 km 2 ), the  Hawaiian Islands  have the most diverse collection of drosophilid flies in the world, living from  rainforests  to  subalpine meadows . These roughly 800 Hawaiian drosophilid species are usually assigned to two genera,  Drosophila  and  Scaptomyza , in the family  Drosophilidae .


The polytene banding of the 'picture wing' group, the best-studied group of Hawaiian drosophilids, enabled Carson to work out the evolutionary tree long before genome analysis was practicable. In a sense, gene arrangements are visible in the banding patterns of each chromosome. Chromosome rearrangements, especially  inversions , make it possible to see which species are closely related.


The results are clear. The inversions, when plotted in tree form (and independent of all other information), show a clear "flow" of species from older to newer islands. There are also cases of colonization back to older islands, and skipping of islands, but these are much less frequent. Using  K-Ar  dating, the present islands date from 0.4 million years ago (mya) ( Mauna Kea ) to 10mya ( Necker ). The oldest member of the Hawaiian archipelago still above the sea is  Kure Atoll , which can be dated to 30 mya. The archipelago itself (produced by the  Pacific Plate  moving over a  hot spot ) has existed for far longer, at least into the  Cretaceous . Previous islands now beneath the sea ( guyots ) form the  Emperor Seamount Chain . [ 54 ]


All of the native  Drosophila  and  Scaptomyza  species in Hawaiʻi have apparently descended from a single ancestral species that colonized the islands, probably 20 million years ago. The subsequent  adaptive radiation  was spurred by a lack of  competition  and a wide variety of  niches . Although it would be possible for a single  gravid  female to colonise an island, it is more likely to have been a group from the same species. [ 55 ] [ 56 ] [ 57 ] [ 58 ]


There are other animals and plants on the Hawaiian archipelago which have undergone similar, if less spectacular, adaptive radiations. [ 59 ] [ 60 ]


Chromosomes display a banded pattern when treated with some stains. Bands are alternating light and dark stripes that appear along the lengths of chromosomes. Unique banding patterns are used to identify chromosomes and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication, translocation or inverted segments. A range of different chromosome treatments produce a range of banding patterns: G-bands, R-bands, C-bands, Q-bands, T-bands and NOR-bands.


Cytogenetics  employs several techniques to visualize different aspects of chromosomes: [ 9 ]


In the "classic" (depicted) karyotype, a  dye , often  Giemsa   (G-banding) , less frequently  mepacrine (quinacrine) , is used to stain bands on the chromosomes. Giemsa is specific for the  phosphate  groups of  DNA . Quinacrine binds to the  adenine - thymine -rich regions. Each chromosome has a characteristic banding pattern that helps to identify them; both chromosomes in a pair will have the same banding pattern.


Karyotypes are arranged with the short arm of the chromosome on top, and the long arm on the bottom.  Some karyotypes call the short and long arms  p  and  q , respectively.  In addition, the differently stained regions and sub-regions are given numerical designations from  proximal  to  distal  on the chromosome arms.  For example,  Cri du chat  syndrome involves a deletion on the short arm of chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is deletion of p15.2 (the  locus  on the chromosome), which is written as 46,XX,del(5)(p15.2). [ 62 ]


Multicolor  FISH  and the older spectral karyotyping are molecular  cytogenetic  techniques used to simultaneously visualize all the pairs of  chromosomes  in an organism in different colors.  Fluorescently  labeled probes for each chromosome are made by labeling chromosome-specific DNA with different  fluorophores . Because there are a limited number of spectrally distinct fluorophores, a combinatorial labeling method is used to generate many different colors. Fluorophore combinations are captured and analyzed by a fluorescence microscope using up to 7 narrow-banded fluorescence filters or, in the case of spectral karyotyping, by using an  interferometer  attached to a fluorescence microscope. In the case of an mFISH image, every combination of fluorochromes from the resulting original images is replaced by a  pseudo color  in a dedicated image analysis software. Thus, chromosomes or chromosome sections can be visualized and identified, allowing for the analysis of chromosomal rearrangements. [ 63 ]
In the case of spectral karyotyping, image processing software assigns a  pseudo color  to each spectrally different combination, allowing the visualization of the individually colored chromosomes. [ 64 ]


Multicolor FISH is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.


Digital karyotyping  is a technique used to quantify the DNA copy number on a genomic scale. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. [ 65 ]  This method is also known as  virtual karyotyping . Using this technique, it is possible to detect small alterations in the human genome, that cannot be detected through methods employing metaphase chromosomes. Some loci deletions are known to be related to the development of cancer. Such deletions are found through digital karyotyping using the loci associated with cancer development. [ 66 ]


Chromosome abnormalities can be numerical, as in the presence of extra or missing chromosomes, or structural, as in  derivative chromosome ,  translocations ,  inversions , large-scale deletions or duplications. Numerical abnormalities, also known as  aneuploidy , often occur as a result of  nondisjunction  during  meiosis  in the formation of a  gamete ;  trisomies , in which three copies of a chromosome are present instead of the usual two, are common numerical abnormalities. Structural abnormalities often arise from errors in  homologous recombination . Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body, or they can occur during  mitosis  and give rise to a  genetic mosaic  individual who has some normal and some abnormal cells.


Chromosomal abnormalities that lead to disease in humans include


Some disorders arise from loss of just a piece of one chromosome, including


Chromosomes were first observed in plant cells by  Carl Wilhelm von Nägeli  in 1842. Their behavior in animal ( salamander ) cells was described by  Walther Flemming , the discoverer of  mitosis , in 1882. The name was coined by another German anatomist,  Heinrich von Waldeyer  in 1888. It is  Neo-Latin  from  Ancient Greek  κάρυον  karyon , "kernel", "seed", or "nucleus", and τύπος  typos , "general form")


The next stage took place after the development of genetics in the early 20th century, when it was appreciated that chromosomes (that can be observed by karyotype) were the carrier of genes. The term karyotype as defined by the  phenotypic  appearance of the  somatic  chromosomes, in contrast to their  genic  contents was introduced by  Grigory Levitsky  who worked with Lev Delaunay,  Sergei Navashin , and  Nikolai Vavilov . [ 67 ] [ 68 ] [ 69 ] [ 70 ]  The subsequent history of the concept can be followed in the works of  C. D. Darlington [ 71 ]  and  Michael JD White . [ 4 ] [ 13 ]


Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal  diploid  human cell contain? [ 72 ]  In 1912,  Hans von Winiwarter  reported 47 chromosomes in  spermatogonia  and 48 in  oogonia , concluding an  XX/XO   sex determination  mechanism. [ 73 ]   Painter  in 1922 was not certain whether the diploid of humans was 46 or 48, at first favoring 46, [ 74 ]  but revised his opinion from 46 to 48, and he correctly insisted on humans having an  XX/XY  system. [ 75 ]  Considering the techniques of the time, these results were remarkable.


Joe Hin Tjio  working in  Albert Levan 's lab [ 76 ]  found the chromosome count to be 46 using new techniques available at the time:


The work took place in 1955, and was published in 1956. The karyotype of humans includes only 46 chromosomes. [ 77 ] [ 29 ]  The other  great apes  have 48 chromosomes.  Human chromosome 2  is now known to be a result of an end-to-end fusion of two ancestral ape chromosomes. [ 78 ] [ 79 ]


The  lacUV5  promoter is a mutated  promoter  from the  Escherichia coli   lac operon  which is used in  molecular biology  to drive  gene expression  on a  plasmid .  lacUV5  is very similar to the classical  lac  promoter, containing just 2 base pair mutations in the  -10 hexamer  region, compared to the  lac  promoter. [ 1 ]   LacUV5  is among the most commonly used promoters in molecular biology because it requires no additional activators and it drives high levels of gene expression. [ 2 ]


The  lacUV5  promoter sequence conforms more closely to the  consensus sequence  recognized by bacterial  sigma factors  than the traditional  lac  promoter does. [ 3 ] : 4b    Due to this,  lacUV5  recruits  RNA Polymerase  more effectively, thus leading to higher transcription of target genes. Additionally, unlike the  lac  promoter,  lacUV5  works independently of  activator  proteins or other  cis regulatory elements  (apart from the -10 and -35 promoter regions). [ 2 ]  While no activators are required,  lacUV5  promoter expression can be regulated by the  LacI repressor  and can be induced with  IPTG , which is an effective inducer of protein expression when used in the concentration range of 100 μM to 1.5 mM. Due to this control, the  lacUV5  promoter is commonly found on expression plasmids and is used when controllable but high levels of a product are desired. [ citation needed ]


The  lacUV5  mutation was first identified in 1970 in a study of  lac  promoter mutants that produce higher yields. Some of them, including UV5, has lost catabolite repression at the CAP site. [ 4 ]  Development into cloning vectors is known since 1982, when a UV5-carrying phage known as "λ h80  lac UV5 cI857" has its genome spliced with the  HaeIII   restriction enzyme  to make plasmids carrying the fragment with UV5. [ 5 ]


Modern  lacUV5  is seen in the BL21(DE3) strain, which carries both a  lac  operon with the standard promoter and a  lacUV5  operon split by the DE3 prophage (and as a result driving the  T7 RNA polymerase  instead). [ 1 ]  The two important mutations are underlined.


Enterobacteria phage λ  ( lambda phage ,  coliphage λ , officially  Escherichia virus Lambda ) is a bacterial virus, or  bacteriophage , that infects the bacterial species  Escherichia coli  ( E. coli ).  It was discovered by  Esther Lederberg  in 1950. [ 2 ]  The wild type of this virus has a  temperate  life cycle that allows it to either reside within the  genome  of its host through  lysogeny  or enter into a  lytic  phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell. [ 3 ]


The phage particle consists of a head (also known as a  capsid ), [ 4 ]  a tail, and tail fibers (see image of virus below). The head contains the phage's double-strand linear  DNA  genome. During infections, the phage particle recognizes and binds to its host,  E. coli , causing DNA in the head of the phage to be ejected through the tail into the cytoplasm of the bacterial cell. Usually, a " lytic cycle " ensues, where the lambda DNA is replicated and new phage particles are produced within the cell. This is followed by cell  lysis , releasing the cell contents, including virions that have been assembled, into the environment. However, under certain conditions, the phage DNA may integrate itself into the host cell chromosome in the  lysogenic  pathway. In this state, the λ DNA is called a  prophage  and stays resident within the host's  genome  without apparent harm to the host. The host is  termed a  lysogen  when a prophage is present. This prophage may enter the lytic cycle when the lysogen enters a stressed condition.


The virus particle consists of a head and a tail that can have tail fibers. The whole particle consists of 12–14 different proteins with more than 1000 protein molecules total and one DNA molecule located in the phage head. However, it is still not entirely clear whether the L and M proteins are part of the virion. [ 5 ]  All characterized lambdoid phages possess an N protein-mediated transcription antitermination mechanism, with the exception of phage HK022. [ 6 ]


The  genome  contains 48,502 [ 7 ]  base pairs of double-stranded, linear DNA, with 12-base single-strand segments at both 5' ends. [ 8 ]  These two single-stranded segments are the "sticky ends" of what is called the  cos  site. The  cos  site circularizes the DNA in the host cytoplasm. In its circular form, the phage genome, therefore, is 48,502 base pairs in length. [ 8 ]   The lambda genome can be inserted into the   E. coli  chromosome and is then called a prophage. See section below for details.


The tail of lambda phages is made of at least 6 proteins (H, J, U, V, Stf, Tfa) and requires 7 more for assembly (I, K, L, M, Z, G/T). This assembly process begins with protein J, which then recruits proteins I, L, K, and G/T to add protein H. Once G and G/T leave the complex, protein V can assemble onto the J/H scaffold. Then, protein U is added to the head-proximal end of the tail. Protein Z is able to connect the tail to the head. Protein H is cleaved due to the actions of proteins U and Z. [ 5 ]


Lambda phage is a non-contractile tailed phage, meaning during an infection event it cannot 'force' its DNA through a bacterial cell membrane. It must instead use an existing pathway to invade the host cell, having evolved the tip of its tail to interact with a specific pore to allow entry of its DNA to the hosts.


On initial infection, the stability of  cII  determines the lifestyle of the phage; stable cII will lead to the lysogenic pathway, whereas if  cII  is degraded the phage will go into the lytic pathway. Low temperature, starvation of the cells and high  multiplicity of infection  (MOI) are known to favor lysogeny (see later discussion). [ 13 ]


This occurs without the N protein interacting with the DNA; the protein instead binds to the freshly transcribed mRNA. Nut sites contain 3 conserved "boxes", of which only BoxB is essential.


This is the lifecycle that the phage follows following most infections, where the cII protein does not reach a high enough concentration due to degradation, so does not activate its promoters. [ citation needed ]


Rightward transcription expresses the  O ,  P  and  Q  genes. O and P are responsible for initiating replication, and Q is another antiterminator that allows the expression of head, tail, and lysis genes from  P R’ . [ 6 ]


Pr is the promoter for rightward transcription, and the cro gene is a regulator gene. The cro gene will encode for the Cro protein that will then repress Prm promoter.  Once Pr transcription is underway the Q gene will then be transcribed at the far end of the operon for rightward transcription. The Q gene is a regulator gene found on this operon, which will control the expression of later genes for rightward transcription. Once the gene's regulatory proteins allow for expression, the Q protein will then act as an anti-terminator. This will then allow for the rest of the operon to be read through until it reaches the transcription terminator. Thus allowing expression of later genes in the operon, and leading to the expression of the lytic cycle. [ 15 ]


Pr promoter has been found to activate the origin in the use of rightward transcription, but the whole picture of this is still somewhat misunderstood. Given there are some caveats to this, for instance this process is different for other phages such as N15 phage, which may encode for DNA polymerase. Another example is the P22 phage may replace the p gene, which encodes for an essential replication protein for something that is capable of encoding for a DnaB helices. [ 6 ]


Q is similar to N in its effect: Q binds to  RNA polymerase  in  Qut  sites and the resulting complex can ignore terminators, however the mechanism is very different; the Q protein first associates with a DNA sequence rather than an mRNA sequence. [ 16 ]


Leftward transcription expresses the  gam,   xis ,  bar  and  int  genes. [ 6 ]  Gam proteins are involved in recombination. Gam is also important in that it inhibits the host RecBCD nuclease from degrading the 3’ ends in rolling circle replication. Int and xis are integration and excision proteins vital to lysogeny. [ citation needed ]


Leftward transcription is believed to result in a deletion mutation of the  rap  gene resulting in a lack of growth of lambda phage. This is due to RNA polymerase attaching to pL promoter site instead of the pR promotor site. Leftward transcription results in  bar I and  bar II transcription on the left operon. Bar  positive phenotype is present when the  rap  gene is absent. The lack of growth of lambda phage is believed to occur due to a temperature sensitivity resulting in inhibition of growth. [ 18 ]