In addition, high fat diets cause chronic low-grade inflammation in the placenta, adipose, liver, brain, and vascular system.  Inflammation  is an important aspect of the bodies’ natural defense system after injury, trauma, or disease.  During an inflammatory response, a series of physiological reactions, such as increased blood flow, increased cellular metabolism, and vasodilation, occur in order to help treat the wounded or infected area.  However, chronic low-grade inflammation has been linked to long-term consequences such as cardiovascular disease, renal failure, aging, diabetes, etc.  This chronic low-grade inflammation is commonly seen in obese individuals on high fat diets.  In a mice model, excessive  cytokines  were detected in mice fed on a high fat diet.  Cytokines aid in cell signaling during immune responses, specifically sending cells towards sites of inflammation, infection, or trauma.  The mRNA of proinflammatory cytokines was induced in the placenta of mothers on high fat diets.  The high fat diets also caused changes in microbiotic composition, which led to hyperinflammatory colonic responses in offspring.  This hyperinflammatory response can lead to  inflammatory bowel diseases  such as  Crohn's disease  or  ulcerative colitis .[35]  As previously mentioned, high fat diets in utero contribute to obesity; however, some proinflammatory factors, like IL-6 and MCP-1, are also linked to body fat deposition.  It has been suggested that histone acetylation is closely associated with inflammation because the addition of  histone deacetylase inhibitors  has been shown to reduce the expression of proinflammatory mediators in  glial cells .  This reduction in inflammation resulted in improved neural cell function and survival.  This inflammation is also often associated with obesity, cardiovascular disease,  fatty liver , brain damage, as well as preeclampsia and preterm birth.  Although it has been shown that high fat diets induce inflammation, which contribute to all these chronic diseases; it is unclear as to how this inflammation acts as a mediator between diet and chronic disease. [ 36 ]


A study done after the Dutch Hunger Winter of 1944-1945 showed that undernutrition during the early stages of pregnancy are associated with hypomethylation of the  insulin-like growth factor II (IGF2)  gene even after six decades.  These individuals had significantly lower methylation rates as compared to their same sex sibling who had not been conceived during the famine.  A comparison was done with children conceived prior to the famine so that their mothers were nutrient deprived during the later stages of gestation; these children had normal methylation patterns. The IGF2 stands for insulin-like growth factor II; this gene is a key contributor in human growth and development.  IGF2 gene is also maternally  imprinted  meaning that the mother's gene is silenced.  The mother's gene is typically methylated at the differentially methylated region (DMR); however, when hypomethylated, the gene is bi-allelically expressed.  Thus, individuals with lower methylation states likely lost some of the imprinting effect.  Similar results have been demonstrated in the Nr3c1 and Ppara genes of the offspring of rats fed on an isocaloric protein-deficient diet before starting pregnancy.  This further implies that the undernutrition was the cause of the epigenetic changes.  Surprisingly, there was not a correlation between methylation states and birth weight.  This displayed that birth weight may not be an adequate way to determine nutritional status during gestation.  This study stressed that epigenetic effects vary depending on the timing of exposure and that early stages of mammalian development are crucial periods for establishing epigenetic marks.  Those exposed earlier in gestation had decreased methylation while those who were exposed at the end of gestation had relatively normal methylation levels. [ 37 ]   The offspring and descendants of mothers with hypomethylation were more likely to develop cardiovascular disease.  Epigenetic alterations that occur during embryogenesis and early fetal development have greater physiologic and metabolic effects because they are transmitted over more mitotic divisions.  In other words, the epigenetic changes that occur earlier are more likely to persist in more cells. [ 37 ]


In another study, researchers discovered that perinatal nutrient restriction resulting in  intrauterine growth restriction  (IUGR) contributes to  diabetes mellitus type 2  (DM2).  IUGR refers to the poor growth of the baby in utero.  In the pancreas, IUGR caused a reduction in the expression of the promoter of the gene encoding a critical transcription factor for beta cell function and development.  Pancreatic beta cells are responsible for making insulin; decreased beta cell activity is associated with DM2 in adulthood.  In skeletal muscle, IUGR caused a decrease in expression of the Glut-4 gene.  The Glut-4 gene controls the production of the Glut-4 transporter; this transporter is specifically sensitive to insulin.  Thus, when insulin levels rise, more glut-4 transporters are brought to the cell membrane to increase the uptake of glucose into the cell.  This change is caused by histone modifications in the cells of skeletal muscle that decrease the effectiveness of the glucose transport system into the muscle. Because the main glucose transporters are not operating at optimal capacity, these individuals are more likely to develop  insulin resistance  with energy rich diets later in life, contributing to DM2. [ 38 ]


Further studies have examined the epigenetic changes resulting from a high protein/low carbohydrate diet during pregnancy.  This diet caused epigenetic changes that were associated with higher blood pressure, higher  cortisol  levels, and a heightened  Hypothalamic-pituitary-adrenal (HPA) axis  response to stress.  Increased methylation in the 11β-hydroxysteroid dehydrogenase type 2 (HSD2),  glucocorticoid receptor (GR) , and  H19  ICR were positively correlated with adiposity and blood pressure in adulthood.  Glucocorticoids play a vital role in tissue development and maturation as well as having effects on metabolism.  Glucocorticoids’ access to GR is regulated by HSD1 and HSD2.  H19 is an imprinted gene for a  long coding RNA (lncRNA) , which has limiting effects on body weight and cell proliferation.  Therefore, higher methylation rates in H19 ICR repress transcription and prevent the lncRNA from regulating body weight.  Mothers who reported higher meat/fish and vegetable intake and lower bread/potato intake in late pregnancy had a higher average methylation in GR and HSD2.  However, one common challenge of these types of studies is that many epigenetic modifications have tissue and cell-type specificity DNA methylation patterns.  Thus, epigenetic modification patterns of accessible tissues, like peripheral blood, may not represent the epigenetic patterns of the tissue involved in a particular disease. [ 39 ]


Strong evidence in rats supports the conclusion that neonatal  estrogen  exposure plays a role in the development of  prostate cancer . Using a human fetal prostate xenograft model, researchers studied the effects of early exposure to estrogen with and without secondary estrogen and testosterone treatment.  A  xenograft model  is a graft of tissue transplanted between organisms of different species.  In this case, human tissue was transplanted into rats; therefore, there was no need to extrapolate from rodents to humans.  Histopathological lesions, proliferation, and serum hormone levels were measured at various time-points after xenografting. At day 200, the xenograft that had been exposed to two treatments of estrogen showed the most severe changes. Additionally, researchers looked at key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors, and tumor suppressors using a custom PCR array. Analysis of DNA methylation showed methylation differences in CpG sites of the stromal compartment after estrogen treatment. These variations in methylation are likely a contributing cause to the changes in the cellular events in the KEGG prostate cancer pathway that inhibit apoptosis and increase cell cycle progression that contribute to the development of cancer. [ 40 ]


In utero or neonatal exposure to  bisphenol A (BPA) , a chemical used in manufacturing polycarbonate plastic, is correlated with higher body weight, breast cancer, prostate cancer, and an altered reproductive function. In a mice model, the mice fed on a BPA diet were more likely to have a yellow coat corresponding to their lower methylation state in the promoter regions of the retrotransposon upstream of the Agouti gene.  The Agouti gene is responsible for determining whether an animal's coat will be banded (agouti) or solid (non-agouti).  However, supplementation with methyl donors like folic acid or phytoestrogen abolished the hypomethylating effect.  This demonstrates that the epigenetic changes can be reversed through diet and supplementation. [ 41 ]


Maternal dietary effects are not just seen in humans, but throughout many taxa in the animal kingdom. These maternal dietary effects can result in ecological changes on a larger scale throughout populations and from generation to generation. The plasticity involved in these epigenetic changes due to maternal diet represents the environment into which the offspring will be born. Many times, epigenetic effects on offspring from the maternal diet during development will genetically prepare the offspring to be better adapted for the environment in which they will first encounter. The epigenetic effects of maternal diet can be seen in many species, utilizing different ecological cues and epigenetic mechanisms to provide an adaptive advantage to future generations.


Within the field of ecology, there are many examples of maternal dietary effects. Unfortunately, the epigenetic mechanisms underlying these phenotypic changes are rarely investigated. In the future, it would be beneficial for ecological scientists as well as epigenetic and genomic scientists to work together to fill the holes within the ecology field to produce a complete picture of environmental cues and epigenetic alterations producing phenotypic diversity.


A  pyralid moth species ,  Plodia interpunctella , commonly found in food storage areas, exhibits maternal dietary effects, as well as paternal dietary effects, on its offspring. Epigenetic changes in moth offspring affect the production of phenoloxidase, an enzyme involved with melanization and correlated with resistance of certain pathogens in many invertebrate species. In this study, parent moths were housed in food rich or food poor environments during their reproductive period. Moths who were housed in food poor environments produced offspring with less phenoloxidase, and thus had a weaker immune system, than moths who reproduced in food rich environments. This is believed to be adaptive because the offspring develop while receiving cues of scarce nutritional opportunities. These cues allow the moth to allocate energy differentially, decreasing energy allocated for the immune system and devoting more energy towards growth and reproduction to increase fitness and insure future generations. One explanation for this effect may be imprinting, the expression of only one parental gene over the other, but further research has yet to be done. [ 42 ]


Parental-mediated dietary epigenetic effects on immunity has a broader significance on wild organisms. Changes in immunity throughout an entire population may make the population more susceptible to an environmental disturbance, such as the introduction of a pathogen. Therefore, these transgenerational epigenetic effects can influence the population dynamics by decreasing the stability of populations who inhabit environments different from the parental environment that offspring are epigenetically modified for.


Food availability also influences the epigenetic mechanisms driving growth rate in the  mouthbrooding cichlid ,  Simochromis pleurospilus . When nutrient availability is high, reproducing females will produce many small eggs, versus fewer, larger eggs in nutrient poor environments. Egg size often correlates with fish larvae body size at hatching: smaller larvae hatch from smaller eggs. In the case of the cichlid, small larvae grow at a faster rate than their larger egg counterparts. This is due to the increased expression of GHR, the growth hormone receptor. Increased transcription levels of GHR genes increase the receptors available to bind with  growth hormone , GH, leading to an increased growth rate in smaller fish. Fish of larger size are less likely to be eaten by predators, therefore it is advantageous to grow quickly in early life stages to insure survival. The mechanism by which GHR transcription is regulated is unknown, but it may be due to hormones within the yolk produced by the mother, or just by the yolk quantity itself. This may lead to DNA methylation or histone modifications which control genic transcription levels. [ 43 ]


Ecologically, this is an example of the mother utilizing her environment and determining the best method to maximize offspring survival, without actually making a conscious effort to do so. Ecology is generally driven by the ability of an organism to compete to obtain nutrients and successfully reproduce. If a mother is able to gather a plentiful amount of resources, she will have a higher fecundity and produce offspring who are able to grow quickly to avoid predation. Mothers who are unable to obtain as many nutrients will produce fewer offspring, but the offspring will be larger in hopes that their large size will help insure survival into sexual maturation. Unlike the moth example, the maternal effects provided to the cichlid offspring do not prepare the cichlids for the environment that they will be born into; this is because mouth brooding cichlids provide parental care to their offspring, providing a stable environment for the offspring to develop. Offspring who have a greater growth rate can become independent more quickly than slow growing counterparts, therefore decreasing the amount of energy spent by the parents during the parental care period.


A similar phenomenon occurs in the  sea urchin ,  Strongylocentrotus droebachiensis . Urchin mothers in nutrient rich environments produce a large number of small eggs. Offspring from these small eggs grow at a faster rate than their large egg counterparts from nutrient poor mothers. Again, it is beneficial for sea urchin larvae, known as  planula , to grow quickly to decrease the duration of their larval phase and metamorphose into a juvenile to decrease predation risks. Sea urchin larvae have the ability to develop into one of two phenotypes, based on their maternal and larval nutrition. Larvae who grow at a fast rate from high nutrition, are able to devote more of their energy towards development into the juvenile phenotype. Larvae who grow at a slower rate with low nutrition, devote more energy towards growing spine-like appendages to protect themselves from predators in an attempt to increase survival into the juvenile phase. The determination of these phenotypes is based on both the maternal and the juvenile nutrition. The epigenetic mechanisms behind these phenotypic changes is unknown, but it is believed that there may be a nutritional threshold that triggers epigenetic changes affecting development and, ultimately, the larval phenotype. [ 44 ]


Meiotic drive  is a type of  intragenomic conflict , whereby one or more  loci  within a  genome  will affect a manipulation of the  meiotic  process in such a way as to favor the transmission of one or more  alleles  over another, regardless of its phenotypic expression.  More simply, meiotic drive is when one copy of a gene is passed on to offspring more than the expected 50% of the time. According to Buckler et al., "Meiotic drive is the subversion of meiosis so that particular genes are preferentially transmitted to the progeny. Meiotic drive generally causes the preferential segregation of small regions of the genome". [ 1 ]


The first report of meiotic drive came from  Marcus Rhoades  who in 1942 observed a violation of  Mendelian segregation  ratios for the R locus - a  gene  controlling the production of the purple pigment  anthocyanin  in  maize  kernels - in a maize line carrying abnormal chromosome 10 (Ab10). [ 2 ]  Ab10 differs from the normal chromosome 10 by the presence of a 150-base pair  heterochromatic  region called 'knob', which functions as a  centromere  during division (hence called 'neocentromere') and moves to the spindle poles faster than the centromeres during  meiosis  I and II. [ 3 ]  The mechanism for this was later found to involve the activity of a  kinesin -14 gene called Kinesin driver ( Kindr ). Kindr protein is a functional  minus-end directed  motor, displaying quicker minus-end directed motility than an endogenous kinesin-14, such as Kin11. As a result  Kindr  outperforms the endogenous kinesins, pulling the 150 bp knobs to the poles faster than the centromeres and causing Ab10 to be preferentially inherited during meiosis  [ 4 ]


The unequal inheritance of gametes has been observed since the 1950s, [ 5 ]  in contrast to  Gregor Mendel 's First and Second Laws (the  law of segregation  and the  law of independent assortment ), which dictate that there is a random chance of each allele being passed on to offspring. Examples of selfish drive genes in animals have primarily been found in rodents and flies. These drive systems could play important roles in the process of  speciation . For instance, the proposal that hybrid sterility ( Haldane's rule ) may arise from the divergent evolution of sex chromosome drivers and their suppressors. [ 6 ]


Early observations of mouse t-haplotypes by Mary Lyon described numerous genetic loci on chromosome 17 that suppress X-chromosome sex ratio distortion. [ 7 ] [ 8 ]  If a driver is left unchecked, this may lead to population extinction as the population would fix for the driver (e.g. a selfish X chromosome), removing the Y chromosome (and therefore males) from the population. The idea that meiotic drivers and their suppressors may govern speciation is supported by observations that mouse Y chromosomes lacking certain genetic loci produce female-biased offspring, implying these loci encode suppressors of drive. [ 9 ]  Moreover, matings of certain mouse strains used in research results in unequal offspring ratios. One gene responsible for sex ratio distortion in mice is r2d2 ( r2d2  – responder to meiotic drive 2), which predicts which strains of mice can successfully breed without offspring sex ratio distortion. [ 10 ]


Selfish chromosomes of  stalk-eyed flies  have had ecological consequences. Driving X chromosomes lead to reductions in male fecundity and mating success, leading to  frequency dependent selection  maintaining both the driving alleles and wild-type alleles. [ 11 ]


Multiple species of fruit fly are known to have driving X chromosomes, of which the best-characterized are found in  Drosophila simulans . Three independent driving X chromosomes are known in  D. simulans , called Paris, Durham, and Winters. In Paris, the driving gene encodes a DNA modelling protein ("heterochromatin protein 1 D2" or  HP1D2 ), where the allele of the driving copy fails to prepare the male Y chromosome for meiosis. [ 12 ]  In Winters, the gene responsible ("Distorter on the X" or  Dox )  has been identified, though the mechanism by which it acts is still unknown. [ 13 ]  The strong selective pressure imposed by these driving X chromosomes has given rise to suppressors of drive, of which the genes are somewhat known for Winters, Durham, and Paris. These suppressors encode hairpin RNAs which match the sequence of driver genes (such as  Dox ), leading host  RNA interference  pathways to degrade  Dox  sequence. [ 14 ]  Autosomal suppressors of drive are known in  Drosophila mediopunctata , [ 15 ]   Drosophila paramelanica , [ 16 ]   Drosophila quinaria , [ 17 ]  and  Drosophila testacea , [ 18 ]  emphasizing the importance of these drive systems in natural populations.


Memetics  is a theory of the evolution of culture based on Darwinian principles with the  meme  as the unit of culture. The term "meme" was coined by biologist  Richard Dawkins  in his 1976 book  The Selfish Gene , [ 1 ]  to illustrate the principle that he later called " Universal Darwinism ". All evolutionary processes depend on  information  being copied, varied, and selected, a process also known as variation with  selective retention . The conveyor of the information being copied is known as the replicator, with the  gene  functioning as the replicator in biological  evolution . Dawkins proposed that the same process drives  cultural evolution , and he called this second replicator the "meme," citing examples such as musical tunes, catchphrases,  fashions , and  technologies . Like genes, memes are selfish replicators and have causal efficacy; in other words, their properties influence their chances of being copied and passed on. Some succeed because they are valuable or useful to their human hosts while others are more like viruses.


Just as genes can work together to form  co-adapted  gene complexes, so groups of memes acting together form co-adapted meme complexes or  memeplexes . Memeplexes include (among many other things)  languages ,  traditions ,  scientific theories ,  financial institutions , and  religions . Dawkins famously referred to religions as " viruses of the mind ". [ 2 ]


Among proponents of memetics are psychologist  Susan Blackmore , author of  The Meme Machine , who argues that when our ancestors began imitating behaviours, they let loose a second replicator and co-evolved to become the "meme machines" that copy, vary, and select memes in culture. [ 3 ]  Philosopher  Daniel Dennett  develops memetics extensively, notably in his books  Darwin's Dangerous Idea , [ 4 ]  and  From Bacteria to Bach and Back . [ 5 ]  He describes the units of memes as "the smallest elements that replicate themselves with reliability and fecundity," [ 6 ]  and claims that "Human consciousness is itself a huge complex of memes." [ 7 ]  In  The Beginning of Infinity , [ 8 ]  physicist  David Deutsch  contrasts static societies that depend on anti-rational memes suppressing innovation and creativity, with dynamic societies based on rational memes that encourage  enlightenment  values, scientific curiosity, and progress.


Criticisms of memetics include claims that memes do not exist, that the analogy with genes is false, that the units cannot be specified, that culture does not evolve through imitation, and that the sources of variation are intelligently designed rather than random. Critics of memetics include biologist  Stephen Jay Gould  who calls memetics a "meaningless metaphor". Philosopher  Dan Sperber  argues against memetics as a viable approach to cultural evolution because cultural items are not directly copied or imitated but are reproduced. [ 9 ]  Anthropologist  Robert Boyd  and biologist  Peter Richerson  work within the alternative, and more mainstream, field of  cultural evolution theory  and  gene-culture coevolution . [ 10 ]   Dual inheritance theory  has much in common with memetics but rejects the idea that memes are replicators. From this perspective, memetics is seen as just one of several approaches to cultural evolution and one that is generally considered less useful than the alternatives of gene-culture coevolution or dual inheritance theory. The main difference is that dual inheritance theory ultimately depends on biological advantage to genes, whereas memetics treats memes as a second replicator in its own right. Memetics also extends to the analysis of  Internet culture  and  Internet memes . [ 11 ]


In his book  The Selfish Gene  (1976), the evolutionary biologist  Richard Dawkins  used the term  meme  to describe a unit of human  cultural transmission  analogous to the  gene , arguing that replication also happens in  culture , albeit in a different sense. While  cultural evolution  itself is a much older topic, with a history that dates back at least as far as  Darwin 's era, Dawkins (1976) proposed that the meme is a unit of culture residing in the brain and is the mutating  replicator  in human cultural evolution. After Dawkins, many discussed this unit of culture as evolutionary "information" which replicates with rules analogous  to  Darwinian selection . [ 12 ]  A  replicator  is a pattern that can influence its surroundings – that is, it has  causal agency  – and can propagate. This proposal resulted in debate among anthropologists, sociologists, biologists, and scientists of other disciplines. Dawkins did not provide a comprehensive explanation of how replication of units of information in the brain controls human behaviour and culture, as the main focus of the book was on gene expression. Dawkins apparently did not intend to present a comprehensive theory of  memetics  in  The Selfish Gene , but rather coined the term  meme  in a speculative spirit. [ citation needed ]  Accordingly, different researchers came to define the term "unit of information" in different ways.


The evolutionary model of cultural information transfer is based on the concept that memes—units of information—have an independent existence, are self-replicating, and are subject to selective evolution through environmental forces. [ 13 ]  Starting from a proposition put forward in the writings of Dawkins, this model has formed the basis of a new area of study, one that looks at the self-replicating units of culture. It has been proposed that just as memes are analogous to genes, memetics is analogous to genetics.


The modern memetics movement dates from the mid-1980s. A January 1983 " Metamagical Themas " column [ 14 ] by  Douglas Hofstadter , in  Scientific American , was influential – as was his 1985 book of the same name. "Memeticist" was coined as analogous to "geneticist" – originally in  The Selfish Gene.  Later Arel Lucas suggested that the discipline that studies memes and their connections to human and other carriers of them be known as "memetics" by analogy with "genetics". [ 14 ]  Dawkins'  The Selfish Gene  has been a factor in attracting the attention of people of disparate intellectual backgrounds. Another stimulus was the publication in 1991 of  Consciousness Explained  by Tufts University philosopher  Daniel Dennett , which incorporated the meme concept into a  theory of the mind . In his 1991 essay " Viruses of the Mind ", Richard Dawkins used memetics to explain the phenomenon of religious belief and the various characteristics of organised religions. By then, memetics had also become a theme appearing in fiction (e.g. Neal Stephenson's  Snow Crash ).


The idea of  language as a virus  had already been introduced by  William S. Burroughs  as early as 1962 in his novel  The Ticket That Exploded , and continued in  The Electronic Revolution , published in 1970 in  The Job .


The foundation of memetics in its full modern incarnation was launched by  Douglas Rushkoff's   Media Virus: Hidden Agendas in Popular Culture  in 1995, [ 15 ]  and was accelerated with the publication in 1996 of two more books by authors outside the academic mainstream:  Virus of the Mind: The New Science of the Meme  by former  Microsoft  executive turned motivational speaker and professional poker-player  Richard Brodie , and  Thought Contagion: How Belief Spreads Through Society  by  Aaron Lynch , a mathematician and philosopher who worked for many years as an engineer at  Fermilab . Lynch claimed to have conceived his theory totally independently of any contact with academics in the cultural evolutionary sphere, and apparently was not aware of  The Selfish Gene  until his book was very close to publication. [ citation needed ]


Around the same time as the publication of the books by Lynch and Brodie the e-journal  Journal of Memetics – Evolutionary Models of Information Transmission [ 16 ]  (published electronically from 1997 to 2005 [ 17 ] ) first appeared. It was first hosted by the Centre for Policy Modelling at  Manchester Metropolitan University . The e-journal soon became the central point for publication and debate within the nascent memeticist community. (There had been a short-lived paper-based memetics publication starting in 1990, the  Journal of Ideas  edited by Elan Moritz. [ 18 ] ) In 1999,  Susan Blackmore , a psychologist at the  University of the West of England , published  The Meme Machine , which more fully worked out the ideas of Dennett, Lynch, and Brodie and attempted to compare and contrast them with various approaches from the cultural evolutionary mainstream, as well as providing novel (and controversial) memetics-based theories for the evolution of language and the human sense of individual selfhood.


The term  meme  derives from the  Ancient Greek  μιμητής ( mimētḗs ), meaning "imitator, pretender". The similar term  mneme  was used in 1904, by the German evolutionary biologist  Richard Semon , best known for his development of the  engram  theory of  memory , in his work  Die mnemischen Empfindungen in ihren Beziehungen zu den Originalempfindungen , translated into English in 1921 as  The Mneme . [ 19 ]  Until  Daniel Schacter  published  Forgotten Ideas, Neglected Pioneers: Richard Semon and the Story of Memory  in 2000, Semon's work had little influence, though it was quoted extensively in  Erwin Schrödinger ’s 1956  Tarner Lecture  “ Mind and Matter ”. Richard Dawkins (1976) apparently coined the word  meme  independently of Semon, writing this:


" 'Mimeme' comes from a suitable Greek root, but I want a monosyllable that sounds a bit like 'gene'. I hope my classicist friends will forgive me if I abbreviate mimeme to meme. If it is any consolation, it could alternatively be thought of as being related to 'memory', or to the French word même." [ 20 ]


David Hull  (2001) pointed out Dawkins's oversight of Semon's work. Hull suggests this early work as an alternative origin to memetics by which Dawkins's memetic theory and classicist connection to the concept can be negotiated.


"Why not date the beginnings of memetics (or mnemetics) as 1904 or at the very least 1914? If [Semon's] two publications are taken as the beginnings of memetics, then the development of memetics [...] has been around for almost a hundred years without much in the way of conceptual or empirical advance!" [ 21 ]


Despite this, Semon's work remains mostly understood as distinct to memetic origins even with the overt similarities accounted for by Hull.


The memetics movement split almost immediately into two. The first group were those who wanted to stick to Dawkins' definition of a meme as "a unit of  cultural transmission ". Gibron Burchett, a memeticist responsible for helping to research and co-coin the term  memetic engineering , along with Leveious Rolando and Larry Lottman, has stated that a meme can be defined, more precisely, as "a unit of  cultural   information  that can be copied, located in the brain". This thinking is more in line with Dawkins' second definition of the meme in his book  The Extended Phenotype . The second group wants to redefine memes as observable  cultural artifacts  and behaviors. However, in contrast to those two positions, the article "Consciousness in meme machines" by Susan Blackmore rejects neither movement. [ 22 ]  Andrej Drapal [ 23 ]  tried to bridge the gap with his differentiation of memes as quantum entities existing per se in quantum superposition and collapsing when detecting by brains from cultural artifacts. Memes are to artifacts as genotypes are to phenotypes.


These two schools became known as the "internalists" and the "externalists." Prominent internalists included both Lynch and Brodie; the most vocal externalists included Derek Gatherer, a geneticist from  Liverpool John Moores University , and William Benzon, a writer on cultural evolution and music. The main rationale for externalism was that internal brain entities are not observable, and memetics cannot advance as a science, especially a  quantitative  science, unless it moves its emphasis onto the directly quantifiable aspects of culture. Internalists countered with various arguments: that brain states will eventually be directly observable with advanced technology, that most cultural anthropologists agree that culture is about  beliefs  and not artifacts, or that artifacts cannot be replicators in the same sense as mental entities (or DNA) are replicators. The debate became so heated that a 1998 Symposium on Memetics, organised as part of the 15th International Conference on  Cybernetics , passed a motion calling for an end to definitional debates. McNamara demonstrated in 2011 that functional connectivity profiling using neuroimaging tools enables the observation of the processing of internal memes, "i-memes", in response to external "e-memes". [ 24 ]
This was developed further in a paper "Memetics and Neural Models of Conspiracy Theories" by Duch, where a model of memes as a quasi-stable neural associative memory  attractor network  is proposed, and a formation of  Memeplex  leading to conspiracy theories illustrated with the simulation of a self-organizing network. [ 25 ]


An advanced statement of the internalist school came in 2002 with the publication of  The Electric Meme , by Robert Aunger, an anthropologist from the  University of Cambridge . Aunger also organised a conference in Cambridge in 1999, at which prominent sociologists and anthropologists were able to give their assessment of the progress made in memetics to that date. This resulted in the publication of  Darwinizing Culture: The Status of Memetics as a Science , edited by Aunger and with a foreword by Dennett, in 2001. [ 26 ]


In 2005, the  Journal of Memetics  ceased publication and published a set of articles on the future of memetics. The website states that although "there was to be a relaunch... after several years nothing has happened". [ 27 ]   Susan Blackmore  left the University of the West of England to become a freelance science-writer and now concentrates more on the field of consciousness and cognitive science. Derek Gatherer moved to work as a computer programmer in the pharmaceutical industry, although he still occasionally publishes on memetics-related matters.  Richard Brodie  is now climbing the world professional poker rankings.  Aaron Lynch  disowned the memetics community and the words "meme" and "memetics" (without disowning the ideas in his book), adopting the self-description "thought contagionist". He died in 2005.


Susan Blackmore  (2002) re-stated the definition of meme as: whatever is copied from one person to another person, whether habits, skills, songs, stories, or any other kind of information. Further she said that memes, like genes, are replicators in the sense as defined by Dawkins. [ 28 ]
That is, they are information that is copied. Memes are copied by  imitation , teaching and other methods. The copies are not perfect: memes are copied with variation; moreover, they compete for space in our memories and for the chance to be copied again. Only some of the variants can survive. The combination of these three elements (copies; variation; competition for survival) forms precisely the condition for  Darwinian evolution , and so memes (and hence human cultures) evolve. Large groups of memes that are copied and passed on together are called  co-adapted  meme complexes, or  memeplexes . In Blackmore's definition, the way that a meme replicates is through imitation. This requires  brain  capacity to generally imitate a model or selectively imitate the model. Since the process of social learning varies from one person to another, the imitation process cannot be said to be completely imitated. The sameness of an idea may be expressed with different memes supporting it. This is to say that the  mutation  rate in memetic evolution is extremely high, and mutations are even possible within each and every iteration of the imitation process. It becomes very interesting when we see that a social system composed of a complex network of microinteractions exists, but at the macro level an order emerges to create culture. [ citation needed ]


Many researchers of cultural evolution regard memetic theory of this time a failed paradigm superseded by  dual inheritance theory . [ 29 ]  Others instead suggest it is not superseded but rather holds a small but distinct intellectual space in cultural evolutionary theory. [ 30 ]


A new framework of  Internet Memetics  initially borrowed Blackmore's conceptual developments but is effectively a data-driven approach, focusing on digital artifacts. This was led primarily by conceptual developments Colin Lankshear and Michele Knobel (2006)  [ 31 ]  and  Limor Shifman  and Mike Thelwall (2009). [ 32 ]  Shiman, in particular, followed Susan Blackmore in rejecting the internalist and externalist debate, however did not offer a clear connection to prior evolutionary frameworks. Later in 2014, she rejected the historical relevance of "information" to memetics. Instead of memes being  units of cultural information , she argued information is exclusively delegated to be "the ways in which addressers position themselves in relation to [a meme instance's] text, its linguistic codes, the addressees, and other potential speakers." [ 33 ]  This is what she called  stance,  which is analytically distinguished from the  content  and  form  of her meme. As such, Shifman's developments can be seen as critical to Dawkins's meme, but also as a somewhat distinct conceptualization of the meme as a communicative system dependent on the internet and social media platforms. By introducing memetics as an internet study there has been a rise in empirical research. That is, memetics in this conceptualization has been notably testable by the application of social science methodologies. It has been popular enough that following Lankshear and Knobel's (2019) review of empirical trends, they warn those interested in memetics that theoretical development should not be ignored, concluding that,


"[R]ight now would be a good time for anyone seriously interested in memes to revisit Dawkins’ work in light of how internet memes have evolved over the past three decades and reflect on what most merits careful and conscientious research attention." [ 34 ]


As Lankshear and Knobel show, the Internet Memetic reconceptualization is limited in addressing long-standing memetic theory concerns. It is not clear that existing Internet Memetic theory's departure from conceptual dichotomies between internalist and externalist debate are compatible with most earlier concerns of memetics. Internet Memetics might be understood as a study without an agreed upon theory, as present research tends to focus on empirical developments answering theories of other areas of cultural research. It exists more as a set of distributed studies than a methodology, theory, field, or discipline, with a few exceptions such as Shifman and those closely following her motivating framework.


Critics contend that some of the proponents' assertions are "untested, unsupported or incorrect." [ 13 ]  Most of the history of memetic criticism has been directed at Dawkins' earlier theory of memetics framed in  The Selfish Gene.  There have been some serious criticisms of memetics. Namely, there are a few key points on which most criticisms focus: mentalism, cultural determinism, Darwinian reduction, a lack of academic novelty, and a lack of empirical evidence of memetic mechanisms.


Luis Benitez-Bribiesca points to the lack of memetic mechanisms. He refers to the lack of a  code script  for memes which would suggest a genuine analogy to DNA in genes. He also suggests the meme mutation mechanism is too unstable which would render the evolutionary process chaotic. That is to say that the "unit of information" which traverses across minds is perhaps too flexible in meaning to be a realistic unit. [ 35 ]  As such, he calls memetics "a  pseudoscientific   dogma " and "a dangerous idea that poses a threat to the serious study of  consciousness  and cultural evolution" among other things.


Another criticism points to memetic triviality. That is, some have argued memetics is derivative of more rich areas of study. One of these cases comes from Peircian  semiotics , (e.g., Deacon, [ 36 ]  Kull [ 37 ] ) stating that the concept of meme is a less developed  Sign . Meme is thus described in memetics as a sign without its triadic nature. Charles Sanders Peirce's semiotic theory involves a triadic structure: a sign (a reference to an object), an object (the thing being referred to), and an interpretant (the interpreting actor of a sign).  For Deacon and Kull, the meme is a degenerate sign, which includes only its ability of being copied. Accordingly, in the broadest sense, the objects of copying are memes, whereas the objects of translation and interpretation are signs.


Others have pointed to the fact that memetics reduces genuine social and communicative activity to genetic arguments, and this cannot adequately describe cultural interactions between people. For example,  Henry Jenkins , Joshua Green, and Sam Ford, in their book  Spreadable Media  (2013), criticize Dawkins' idea of the meme, writing that "while the idea of the meme is a compelling one, it may not adequately account for how content circulates through participatory culture." The three authors also criticize other interpretations of memetics, especially those which describe memes as "self-replicating", because they ignore the fact that "culture is a human product and replicates through human agency." [ 38 ]  In doing so, they align more closely with Shifman's notion of Internet Memetics and her addition of the human agency of  stance  to describe participatory structure.


Mary Midgley  criticizes memetics for at least two reasons: [ 39 ]


Like other critics, Maria Kronfeldner has criticized memetics for being based on an allegedly inaccurate analogy with the gene; alternately, she claims it is "heuristically trivial", being a mere redescription of what is already known without offering any useful novelty. [ 41 ]


Research methodologies that apply memetics go by many names:  Viral marketing , cultural evolution, the history of ideas, social analytics, and more.  Many of these applications do not make reference to the literature on memes directly but are built upon the evolutionary lens of idea propagation that treats semantic units of culture as self-replicating and mutating patterns of information that are assumed to be relevant for scientific study.  For example, the field of public relations is filled with attempts to introduce new ideas and alter social discourse.  One means of doing this is to design a meme and deploy it through various media channels.  One historic example of applied memetics is the PR campaign conducted in 1991 as part of the build-up to the first Gulf War in the United States. [ 54 ]


The application of memetics to a difficult complex social system problem, environmental  sustainability , has recently been attempted at thwink.org [ 55 ]  Using meme types and memetic infection in several stock and flow simulation models, Jack Harich has demonstrated several interesting phenomena that are best, and perhaps only, explained by memes. One model, The Dueling Loops of the Political Powerplace, [ 56 ]  argues that the fundamental reason corruption is the norm in politics is due to an inherent structural advantage of one feedback loop pitted against another. Another model, The Memetic Evolution of Solutions to Difficult Problems, [ 57 ]  uses memes, the  evolutionary algorithm , and the  scientific method  to show how complex solutions evolve over time and how that process can be improved. The insights gained from these models are being used to engineer memetic solution elements to the sustainability problem.


Another application of memetics in the sustainability space is the crowdfunded Climate Meme Project [ 58 ]  conducted by Joe Brewer and Balazs Laszlo Karafiath in the spring of 2013. This study was based on a collection of 1000 unique text-based expressions gathered from Twitter, Facebook, and structured interviews with climate activists. The major finding was that the global warming meme is not effective at spreading because it causes emotional duress in the minds of people who learn about it.  Five central tensions were revealed in the discourse about [climate change], each of which represents a resonance point through which dialogue can be engaged.  The tensions were Harmony/Disharmony (whether or not humans are part of the natural world), Survival/Extinction (envisioning the future as either apocalyptic collapse of civilization or total extinction of the human race), Cooperation/Conflict (regarding whether or not humanity can come together to solve global problems), Momentum/Hesitation (about whether or not we are making progress at the collective scale to  address climate change ), and Elitism/Heretic (a general sentiment that each side of the debate considers the experts of its opposition to be untrustworthy). [ 59 ]


Ben Cullen, in his book  Contagious Ideas , [ 60 ]  brought the idea of the meme into the discipline of archaeology. He coined the term "Cultural Virus Theory", and used it to try to anchor archaeological theory in a neo-Darwinian paradigm. Archaeological memetics could assist the application of the meme concept to material culture in particular.


Francis Heylighen  of the  Center Leo Apostel for Interdisciplinary Studies  has postulated what he calls "memetic selection criteria". These criteria opened the way to a specialized field of  applied memetics  to find out if these selection criteria could stand the test of  quantitative analyses . In 2003 Klaas Chielens carried out these tests in a Masters thesis project on the testability of the selection criteria.


In  Selfish Sounds and Linguistic Evolution , [ 61 ]  Austrian linguist Nikolaus Ritt has attempted to operationalise memetic concepts and use them for the explanation of long term sound changes and change conspiracies in early English. It is argued that a generalised Darwinian framework for handling cultural change can provide explanations where established, speaker centred approaches fail to do so. The book makes comparatively concrete suggestions about the possible material structure of memes, and provides two empirically rich case studies.


Australian academic S.J. Whitty has argued that  project management  is a memeplex with the language and stories of its practitioners at its core. [ 62 ]  This radical approach sees a project and its management as an illusion; a human construct about a collection of feelings, expectations, and sensations, which are created, fashioned, and labeled by the human brain. Whitty's approach requires project managers to consider that the reasons for using project management are not consciously driven to maximize profit, and are encouraged to consider project management as naturally occurring, self-serving, evolving process which shapes organizations for its own purpose.


Swedish political scientist Mikael Sandberg argues against " Lamarckian " interpretations of institutional and technological evolution and studies creative innovation of  information technologies  in governmental and private organizations in Sweden in the 1990s from a memetic perspective. [ 63 ]  Comparing the effects of active ("Lamarckian") IT strategy versus user–producer interactivity (Darwinian co-evolution), evidence from Swedish organizations shows that co-evolutionary interactivity is almost four times as strong a factor behind IT creativity as the "Lamarckian" IT strategy.


A  microsatellite  is a tract of repetitive  DNA  in which certain  DNA motifs  (ranging in length from one to six or more  base pairs ) are repeated, typically 5–50 times. [ 1 ] [ 2 ]  Microsatellites occur at thousands of locations within an organism's  genome . They have a higher  mutation  rate than other areas of DNA [ 3 ]  leading to high  genetic diversity . Microsatellites are often referred to as  short tandem repeats  ( STRs ) by  forensic geneticists  and in  genetic genealogy , or as  simple sequence repeats  ( SSRs ) by plant geneticists. [ 4 ]


Microsatellites and their longer cousins, the  minisatellites , together are classified as  VNTR  (variable number of  tandem repeats ) DNA. The name  "satellite" DNA  refers to the early observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying "satellite" layers of repetitive DNA. [ 5 ]


They are widely used for  DNA profiling  in  cancer diagnosis , in  kinship  analysis (especially  paternity testing ) and in forensic identification. They are also used in  genetic linkage  analysis to locate a gene or a mutation responsible for a given trait or disease. Microsatellites are also used in  population genetics  to measure levels of relatedness between subspecies, groups and individuals.


Although the first microsatellite was characterised in 1984 at the  University of Leicester  by Weller,  Jeffreys  and colleagues as a polymorphic GGAT repeat in the human  myoglobin  gene, the term "microsatellite" was introduced later, in 1989, by Litt and Luty. [ 1 ]  The name  "satellite" DNA  refers to the early observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying "satellite" layers of repetitive DNA. [ 5 ]  The increasing availability of DNA amplification by PCR at the beginning of the 1990s triggered a large number of studies using the amplification of microsatellites as genetic markers for forensic medicine, for paternity testing, and for positional cloning to find the gene underlying a trait or disease. Prominent early applications include the identifications by microsatellite genotyping of the eight-year-old skeletal remains of a British murder victim ( Hagelberg  et al. 1991), and of the Auschwitz concentration camp doctor  Josef Mengele  who escaped to South America following World War II ( Jeffreys  et al. 1992). [ 1 ]


A microsatellite is a tract of tandemly repeated (i.e. adjacent) DNA motifs that range in length from one to six or up to ten nucleotides (the exact definition and delineation to the longer minisatellites varies from author to author), [ 1 ] [ 6 ]  and are typically repeated 5–50 times. For example, the sequence TATATATATA is a dinucleotide microsatellite, and GTCGTCGTCGTCGTC is a trinucleotide microsatellite (with A being  Adenine , G  Guanine , C  Cytosine , and T  Thymine ). Repeat units of four and five nucleotides are referred to as tetra- and pentanucleotide motifs, respectively. Most  eukaryotes  have microsatellites, with the notable exception of some yeast species. Microsatellites are distributed throughout the genome. [ 7 ] [ 1 ] [ 8 ]  The human genome for example contains 50,000–100,000 dinucleotide microsatellites, and lesser numbers of tri-, tetra- and pentanucleotide microsatellites. [ 9 ]  Many are located in non-coding parts of the human genome and therefore do not produce proteins, but they can also be located in regulatory regions and  coding regions .


Microsatellites in non-coding regions may not have any specific function, and therefore might not be  selected  against; this allows them to accumulate mutations unhindered over the generations and gives rise to variability that can be used for DNA fingerprinting and identification purposes. Other microsatellites are located in regulatory flanking or  intronic  regions of genes, or directly in  codons  of genes – microsatellite mutations in such cases can lead to phenotypic changes and diseases, notably in  triplet expansion diseases  such as  fragile X syndrome  and  Huntington's disease . [ 10 ]


Telomeres  are linear sequences of DNA that sit at the very ends of chromosomes and protect the integrity of genomic material (not unlike an  aglet  on the end of a shoelace) during successive rounds of cell division due to the "end replication problem". [ 6 ]  In white blood cells, the gradual shortening of telomeric DNA has been shown to inversely correlate with  ageing  in several sample types. [ 11 ]  Telomeres consist of repetitive DNA, with the hexanucleotide repeat motif TTAGGG in vertebrates. [ citation needed ]  They are thus classified as  minisatellites . Similarly, insects have shorter repeat motifs in their telomeres that could arguably be considered microsatellites. [ citation needed ]


Unlike  point mutations , which affect only a single nucleotide, microsatellite mutations lead to the gain or loss of an entire repeat unit, and sometimes two or more repeats simultaneously. Thus, the  mutation rate  at microsatellite  loci  is expected to differ from other mutation rates, such as base substitution rates. [ 12 ] [ 13 ]  The mutation rate at microsatellite loci depends on the repeat motif sequence, the number of repeated motif units and the purity of the canonical repeated sequence. [ 14 ]   A variety of mechanisms for mutation of microsatellite loci have been reviewed, [ 14 ] [ 15 ]   and their resulting polymorphic nature has been quantified. [ 16 ]  The actual cause of mutations in microsatellites is debated.


One proposed cause of such length changes is replication slippage, caused by mismatches between DNA strands while being replicated during  meiosis . [ 17 ]   DNA polymerase , the enzyme responsible for reading DNA during replication, can slip while moving along the template strand and continue at the wrong nucleotide. DNA polymerase slippage is more likely to occur when a repetitive sequence (such as CGCGCG) is replicated. Because microsatellites consist of such repetitive sequences, DNA polymerase may make errors at a higher rate in these sequence regions. Several studies have found evidence that slippage is the cause of microsatellite mutations. [ 18 ] [ 19 ]  Typically, slippage in each microsatellite occurs about once per 1,000 generations. [ 20 ]  Thus, slippage changes in repetitive DNA are three orders of magnitude more common than  point mutations  in other parts of the genome. [ 21 ]  Most slippage results in a change of just one repeat unit, and slippage rates vary for different allele lengths and repeat unit sizes, [ 3 ]  and within different species. [ 22 ] [ 23 ] [ 24 ]  If there is a large size difference between individual alleles, then there may be increased instability during recombination at meiosis. [ 21 ]


Another possible cause of microsatellite mutations are point mutations, where only one nucleotide is incorrectly copied during replication. A study comparing human and primate genomes found that most changes in repeat number in short microsatellites appear due to point mutations rather than slippage. [ 25 ]


Direct estimates of microsatellite mutation rates have been made in numerous organisms, from insects to humans. In the  desert locust   Schistocerca gregaria , the microsatellite mutation rate was estimated at 2.1 × 10 −4  per generation per locus. [ 26 ]  The microsatellite mutation rate in human male germ lines is five to six times higher than in female germ lines and ranges from 0 to 7 × 10 −3  per locus per gamete per generation. [ 3 ]  In the nematode  Pristionchus pacificus , the estimated microsatellite mutation rate ranges from 8.9 × 10 −5  to 7.5 × 10 −4  per locus per generation. [ 27 ]


Microsatellite mutation rates vary with base position relative to the microsatellite, repeat type, and base identity. [ 25 ]  Mutation rate rises specifically with repeat number, peaking around six to eight repeats and then decreasing again. [ 25 ]  Increased heterozygosity in a population will also increase microsatellite mutation rates, [ 28 ]  especially when there is a large length difference between alleles. This is likely due to  homologous chromosomes  with arms of unequal lengths causing instability during meiosis. [ 29 ]


Many microsatellites are located in  non-coding DNA  and are biologically silent. Others are located in regulatory or even  coding DNA  – microsatellite mutations in such cases can lead to phenotypic changes and diseases. A genome-wide study estimates that microsatellite variation contributes 10–15% of heritable gene expression variation in humans. [ 30 ] [ 16 ]


In mammals, 20–40% of proteins contain repeating sequences of  amino acids  encoded by short sequence repeats. [ 31 ]  Most of the short sequence repeats within protein-coding portions of the genome have a repeating unit of three nucleotides, since that length will not cause frame-shifts when mutating. [ 32 ]  Each trinucleotide repeating sequence is transcribed into a repeating series of the same amino acid. In yeasts, the most common repeated amino acids are glutamine, glutamic acid, asparagine, aspartic acid and serine.


Mutations in these repeating segments can affect the physical and chemical properties of proteins, with the potential for producing gradual and predictable changes in protein action. [ 33 ]  For example, length changes in tandemly repeating regions in the  Runx2  gene lead to differences in facial length in domesticated dogs ( Canis familiaris ), with an association between longer sequence lengths and longer faces. [ 34 ]  This association also applies to a wider range of Carnivora species. [ 35 ]  Length changes in polyalanine tracts within the  HOXA13  gene are linked to  hand-foot-genital syndrome , a developmental disorder in humans. [ 36 ]  Length changes in other triplet repeats are linked to more than 40 neurological diseases in humans, notably  trinucleotide repeat disorders  such as  fragile X syndrome  and  Huntington's disease . [ 10 ]  Evolutionary changes from replication slippage also occur in simpler organisms. For example, microsatellite length changes are common within surface membrane proteins in yeast, providing rapid evolution in cell properties. [ 37 ]  Specifically, length changes in the FLO1 gene control the level of adhesion to substrates. [ 38 ]  Short sequence repeats also provide rapid evolutionary change to surface proteins in pathenogenic bacteria; this may allow them to keep up with immunological changes in their hosts. [ 39 ]  Length changes in short sequence repeats in a fungus ( Neurospora crassa ) control the duration of its  circadian clock  cycles. [ 40 ]


Length changes of microsatellites within  promoters  and other  cis-regulatory regions  can change gene expression quickly, between generations. The human genome contains many (>16,000) short sequence repeats in regulatory regions, which provide 'tuning knobs' on the expression of many genes. [ 30 ] [ 41 ]


Length changes in bacterial SSRs can affect  fimbriae  formation in  Haemophilus influenzae , by altering promoter spacing. [ 39 ]  Dinucleotide microsatellites are linked to abundant variation in cis-regulatory control regions in the human genome. [ 41 ]  Microsatellites in control regions of the Vasopressin 1a receptor gene in voles influence their social behavior, and level of monogamy. [ 42 ]


In  Ewing sarcoma  (a type of painful bone cancer in young humans), a point mutation has created an extended GGAA microsatellite which binds a transcription factor, which in turn activates the EGR2 gene which drives the cancer. [ 43 ]  In addition, other GGAA microsatellites may influence the expression of genes that contribute to the clinical outcome of Ewing sarcoma patients. [ 44 ]


Microsatellites within  introns  also influence phenotype, through means that are not currently understood. For example, a GAA triplet expansion in the first intron of the X25 gene appears to interfere with transcription, and causes  Friedreich's ataxia . [ 45 ]  Tandem repeats in the first intron of the Asparagine synthetase gene are linked to acute lymphoblastic leukaemia. [ 46 ]  A repeat polymorphism in the fourth intron of the NOS3 gene is linked to hypertension in a Tunisian population. [ 47 ]  Reduced repeat lengths in the EGFR gene are linked with osteosarcomas. [ 48 ]


An archaic form of splicing preserved in  zebrafish  is known to use microsatellite sequences within intronic mRNA for the removal of introns in the absence of U2AF2 and other splicing machinery. It is theorized that these sequences form highly stable  cloverleaf  configurations that bring the 3' and 5' intron splice sites into close proximity, effectively replacing the  spliceosome . This method of RNA splicing is believed to have diverged from human evolution at the formation of  tetrapods  and to represent an artifact of an  RNA world . [ 49 ]


Almost 50% of the human genome is contained in various types of transposable elements (also called transposons, or 'jumping genes'), and many of them contain repetitive DNA. [ 50 ]  It is probable that short sequence repeats in those locations are also involved in the regulation of gene expression. [ 51 ]


Microsatellites are used for assessing chromosomal DNA deletions in cancer diagnosis. Microsatellites are widely used for  DNA profiling , also known as "genetic fingerprinting", of crime stains (in forensics) and of tissues (in transplant patients).  They are also widely used in  kinship  analysis (most commonly in paternity testing). Also, microsatellites are used for mapping locations  within the genome, specifically in  genetic linkage  analysis to locate a gene or a mutation responsible for a given trait or disease. As a special case of mapping, they can be used for studies of  gene duplication  or  deletion . Researchers use microsatellites in  population genetics  and in species conservation projects. Plant geneticists have proposed the use of microsatellites for  marker assisted selection  of desirable traits in plant breeding.


In  tumour  cells, whose controls on replication are damaged, microsatellites may be gained or lost at an especially high frequency during each round of  mitosis . Hence a tumour cell line might show a different  genetic fingerprint  from that of the host tissue, and, especially in  colorectal cancer , might present with  loss of heterozygosity . [ 52 ] [ 53 ]  Microsatellites analyzed in primary tissue therefore been routinely used in cancer diagnosis to assess tumour progression. [ 54 ] [ 55 ] [ 56 ]  Genome Wide Association Studies (GWAS) have been used to identify microsatellite biomarkers as a source of genetic predisposition in a variety of cancers. [ 57 ] [ 58 ] [ 59 ]


Microsatellite analysis became popular in the field of  forensics  in the 1990s. [ 60 ]  It is used for the  genetic fingerprinting  of individuals where it permits forensic identification (typically matching a crime stain to a victim or perpetrator). It is also used to follow up  bone marrow transplant  patients. [ 61 ]


The microsatellites in use today for forensic analysis are all tetra- or penta-nucleotide repeats, as these give a high degree of error-free data while being short enough to survive degradation in non-ideal conditions.  Even shorter repeat sequences would tend to suffer from artifacts such as PCR stutter and preferential amplification, while longer repeat sequences would suffer more highly from environmental degradation and would amplify less well by  PCR . [ 62 ]  Another forensic consideration is that the person's  medical privacy  must be respected, so that forensic STRs are chosen which are non-coding, do not influence gene regulation, and are not usually trinucleotide STRs which could be involved in  triplet expansion diseases  such as  Huntington's disease . Forensic STR profiles are stored in DNA databanks such as the  UK National DNA Database  (NDNAD), the American  CODIS  or the Australian NCIDD.


Autosomal  microsatellites are widely used for  DNA profiling  in  kinship  analysis (most commonly in paternity testing). [ 63 ]  Paternally inherited  Y-STRs  (microsatellites on the  Y chromosome ) are often used in  genealogical DNA testing .


During the 1990s and the first several years of this millennium, microsatellites were the workhorse genetic markers for genome-wide scans to locate any gene responsible for a given phenotype or disease, using  segregation  observations across generations of a sampled pedigree. Although the rise of higher throughput and cost-effective  single-nucleotide polymorphism  (SNP) platforms led to the era of the SNP for genome scans, microsatellites remain highly informative measures of genomic variation for linkage and association studies. Their continued advantage lies in their greater allelic diversity than biallelic SNPs, thus microsatellites can differentiate alleles within a SNP-defined linkage disequilibrium block of interest. Thus, microsatellites have successfully led to discoveries of type 2 diabetes ( TCF7L2 ) and prostate cancer genes (the 8q21 region). [ 6 ] [ 64 ]


Microsatellites were popularized in  population genetics  during the 1990s because as  PCR  became ubiquitous in laboratories researchers were able to design primers and amplify sets of microsatellites at low cost. Their uses are wide-ranging. [ 66 ]  A microsatellite with a neutral evolutionary history makes it applicable for measuring or inferring  bottlenecks , [ 67 ]   local adaptation , [ 68 ]  the allelic  fixation index  (F ST ), [ 69 ]   population size , [ 70 ]  and  gene flow . [ 71 ]  As  next generation sequencing  becomes more affordable the use of microsatellites has decreased, however they remain a crucial tool in the field. [ 72 ]


Marker assisted selection  or marker aided selection (MAS) is an indirect selection process where a  trait  of interest is selected based on a  marker  ( morphological ,  biochemical  or  DNA / RNA  variation) linked to a trait of interest (e.g. productivity, disease resistance, stress tolerance, and quality), rather than on the trait itself. Microsatellites have been proposed to be used as such markers to assist plant breeding. [ 73 ]


Repetitive DNA is not easily analysed by  next generation DNA sequencing  methods, for some technologies struggle with  homopolymeric  tracts. A variety of software approaches have been created for the analysis or raw nextgen DNA sequencing reads to determine the genotype and variants at repetitive loci. [ 75 ] [ 76 ]  Microsatellites can be analysed and verified by established PCR amplification and amplicon size determination, sometimes followed by  Sanger DNA sequencing .


In forensics, the analysis is performed by extracting  nuclear DNA  from the cells of a sample of interest, then amplifying specific  polymorphic  regions of the extracted DNA by means of the  polymerase chain reaction . Once these sequences have been amplified, they are resolved either through  gel electrophoresis  or  capillary electrophoresis , which will allow the analyst to determine how many repeats of the microsatellites sequence in question there are.  If the DNA was resolved by gel electrophoresis, the DNA can be visualized either by  silver staining  (low sensitivity, safe, inexpensive), or an  intercalating dye  such as  ethidium bromide  (fairly sensitive, moderate health risks, inexpensive), or as most modern forensics labs use,  fluorescent dyes  (highly sensitive, safe, expensive). [ 77 ]  Instruments built to resolve microsatellite fragments by capillary electrophoresis also use fluorescent dyes. [ 77 ]  Forensic profiles are stored in major databanks. The  British  data base for microsatellite loci identification was originally based on the British  SGM+  system [ 78 ] [ 79 ]  using 10 loci and a  sex marker . The Americans [ 80 ]  increased this number to 13 loci. [ 81 ]  The Australian database is called the NCIDD, and since 2013 it has been using 18 core markers for DNA profiling. [ 60 ]


Microsatellites can be amplified for identification by the  polymerase chain reaction  (PCR) process, using the unique sequences of flanking regions as  primers .  DNA is repeatedly denatured at a high temperature to separate the double strand, then cooled to allow  annealing  of primers and the extension of nucleotide sequences through the microsatellite.  This process results in production of enough DNA to be visible on  agarose  or  polyacrylamide  gels; only small amounts of DNA are needed for amplification because in this way thermocycling creates an exponential increase in the replicated segment. [ 82 ]  With the abundance of PCR technology, primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is often a tedious and costly process.


If searching for microsatellite markers in specific regions of a genome, for example within a particular  intron , primers can be designed manually. This involves searching the genomic DNA sequence for microsatellite repeats, which can be done by eye or by using automated tools such as  repeat masker . Once the potentially useful microsatellites are determined, the flanking sequences can be used to design  oligonucleotide  primers which will amplify the specific microsatellite repeat in a PCR reaction.


Random microsatellite primers can be developed by  cloning  random segments of DNA from the focal species. These random segments are inserted into a  plasmid  or  bacteriophage   vector , which is in turn implanted into  Escherichia coli  bacteria. Colonies are then developed, and screened with fluorescently–labelled oligonucleotide sequences that will hybridize to a microsatellite repeat, if present on the DNA segment. If positive clones can be obtained from this procedure, the DNA is sequenced and PCR primers are chosen from sequences flanking such regions to determine a specific  locus . This process involves significant trial and error on the part of researchers, as microsatellite repeat sequences must be predicted and primers that are randomly isolated may not display significant polymorphism. [ 21 ] [ 83 ]  Microsatellite loci are widely distributed throughout the genome and can be isolated from semi-degraded DNA of older specimens, as all that is needed is a suitable substrate for amplification through PCR.


More recent techniques involve using oligonucleotide sequences consisting of repeats complementary to repeats in the microsatellite to "enrich" the DNA extracted ( microsatellite enrichment ). The oligonucleotide probe hybridizes with the repeat in the microsatellite, and the probe/microsatellite complex is then pulled out of solution. The enriched DNA is then cloned as normal, but the proportion of successes will now be much higher, drastically reducing the time required to develop the regions for use. However, which probes to use can be a trial and error process in itself. [ 84 ]


ISSR  (for  inter-simple sequence repeat ) is a general term for a genome region between microsatellite loci. The complementary sequences to two neighboring microsatellites are used as PCR primers; the variable region between them gets amplified. The limited length of amplification cycles during PCR prevents excessive replication of overly long contiguous DNA sequences, so the result will be a mix of a variety of amplified DNA strands which are generally short but vary much in length.


Sequences amplified by ISSR-PCR can be used for DNA fingerprinting. Since an ISSR may be a conserved or nonconserved region, this technique is not useful for distinguishing individuals, but rather for  phylogeography  analyses or maybe delimiting  species ; sequence diversity is lower than in SSR-PCR, but still higher than in actual gene sequences. In addition, microsatellite sequencing and ISSR sequencing are mutually assisting, as one produces primers for the other.


Repetitive DNA is not easily analysed by  next generation DNA sequencing  methods, which struggle with homopolymeric tracts. [ 85 ]  Therefore, microsatellites are normally analysed by conventional PCR amplification and amplicon size determination. The use of PCR means that microsatellite length analysis is prone to PCR limitations like any other PCR-amplified DNA locus. A particular concern is the occurrence of ' null alleles ':


A  minichromosome  is a small  chromatin -like structure resembling a  chromosome  and consisting of  centromeres ,  telomeres  and  replication origins [ 1 ]  but little additional genetic material. [ 2 ] [ self-published source? ]  They replicate autonomously in the cell during  cellular division . [ 3 ]  Minichromosomes may be created by natural processes as chromosomal aberrations or by  genetic engineering . [ 1 ]


Minichromosomes can be either linear or circular pieces of  DNA . [ 3 ]  By minimizing the amount of unnecessary genetic information on the chromosome and including the basic components necessary for  DNA replication  (centromere, telomeres, and replication sequences), molecular biologists aim to construct a chromosomal platform which can be utilized to insert or present new  genes  into a  host cell . [ 3 ]


Producing minichromosomes by genetic engineering techniques involves two primary methods, the  de novo  (bottom-up) and the top-down approach. [ 1 ]


The minimum constituent parts of a chromosome (centromere, telomeres, and DNA replication sequences) are assembled [ 4 ]  by using  molecular cloning  techniques to construct the desired chromosomal contents  in vitro . Next, the desired contents of the minichromosome must be  transformed  into a host which is capable of assembling the components (typically yeast or mammalian cells [ 5 ] ) into a functional chromosome. This approach has been attempted for the introduction of minichromosomes into  maize  for the possibility of genetic engineering, but success has been limited and questionable. [ 6 ]  In general, the  de novo  approach is more difficult than the top-down method due to species incompatibility issues and the  heterochromatic  nature of centromeric regions. [ 5 ]


This method utilizes the mechanism of  telomere -mediated chromosomal truncation (TMCT). This process is the generation of  truncation  by selective transformation of telomeric sequences into a host genome. This insertion causes the generation of more telomeric sequences and eventual truncation. [ 3 ]  The newly synthesized truncated chromosome can then be altered through the insertion of new genes for desired traits. The top-down approach is generally considered as the more plausible means of generating extra-numerary chromosomes for the use of genetic engineering of plants. In particular it is useful because their stability during cell division has been demonstrated. [ 7 ]  The limitation of this approach is that it is labor-intensive.


Unlike traditional methods of genetic engineering, minichromosomes can be used to transfer and express multiple sets of genes onto one engineered chromosome package. [ 8 ]  Traditional methods which involve the insertion of novel genes into existing sequences may result in the disruption of endogenous genes [ 1 ]  and thus negatively affect the host cell. Additionally, with traditional gene insertion methods, scientists have had less ability to control where the newly inserted genes are located on the host cell chromosomes, [ 9 ]  which makes it difficult to predict inheritance of multiple genes from generation to generation. Minichromosome technology allows for the stacking of genes side-by-side on the same chromosome thus reducing likelihood of segregation of novel traits.


In 2006, scientists demonstrated the successful use of telomere truncation in maize plants to produce minichromosomes that could be utilized as a platform for inserting genes into the plant genome. [ 10 ]  In plants, the telomere sequence is conserved, which implies that this strategy can be utilized to successfully construct additional minichromosomes in other plant species. [ 1 ]


In 2007, scientists reported success in assembling minichromosomes  in vitro  using the  de novo  method. [ 6 ]


The use of minichromosomes as a means for generating more desirable crop traits is actively being explored. Major advantages include the ability to introduce genetic information which is highly compatible with the host genome. This eliminates the risk of disrupting various important processes such as cell division and gene expression. With continued development, the future for use of minichromosomes may make a huge impact on the productivity of major crops. [ 11 ]


Minichromosomes have also been successfully inserted into yeast and animal cells. These minichromosomes were constructed using the  de novo  approach. [ 3 ]


The  mobilome  is the entire set of  mobile genetic elements  in a  genome . Mobilomes are found in  eukaryotes , [ 1 ]   prokaryotes , [ 2 ]  and  viruses . [ 3 ]  The compositions of mobilomes differ among lineages of life, with  transposable elements  being the major mobile elements in eukaryotes, and  plasmids  and  prophages  being the major types in prokaryotes. [ 4 ]   Virophages  contribute to the viral mobilome. [ 5 ]


Transposable elements  are elements that can move about or propagate within the genome, and are the major constituents of the  eukaryotic  mobilome. [ 4 ]  Transposable elements can be regarded as genetic  parasites  because they exploit the host  cell's   transcription  and  translation  mechanisms to extract and insert themselves in different parts of the genome, regardless of the  phenotypic  effect on the host. [ 6 ]


Eukaryotic transposable elements were first discovered in  maize  ( Zea mays ) in which  kernels  showed a dotted color pattern. [ 7 ]   Barbara McClintock  described the maize  Ac/Ds system  in which the Ac  locus  promotes the excision of the Ds locus from the genome, and excised Ds elements can  mutate   genes  responsible for  pigment  production by  inserting  into their  coding regions . [ 8 ]


Other examples of transposable elements include:  yeast  ( Saccharomyces cerevisiae )  Ty elements , a  retrotransposon  which encodes a  reverse transcriptase  to convert its  mRNA transcript  into DNA which can then insert into other parts of the genome; [ 9 ] [ 10 ]  and  fruit fly  ( Drosophila melanogaster )  P-elements , which randomly inserts into the genome to cause mutations in  germ line cells , but not in  somatic cells . [ 11 ]


Plasmids were discovered in the 1940s as genetic materials outside of  bacterial   chromosomes . [ 12 ]  Prophages are genomes of  bacteriophages  (a type of virus) that are inserted into bacterial chromosomes; prophages can then be spread to other bacteria through the  lytic cycle  and  lysogenic cycle  of  viral replication . [ 13 ]


While transposable elements are also found in prokaryotic genomes, [ 14 ]  the most common mobile genetic elements in the prokaryotic genome are  plasmids  and  prophages . [ 4 ]


Plasmids and prophages can move between genomes through  bacterial conjugation , allowing  horizontal gene transfer . [ 15 ]  Plasmids often carry genes that are responsible for bacterial  antibiotic resistance ; as these plasmids replicate and pass from one genome to another, the whole bacterial  population  can quickly  adapt  to the  antibiotic . [ 16 ] [ 17 ]  Prophages can loop out of bacterial chromosomes to produce bacteriophages that go on to infect other bacteria with the prophages; this allows prophages to propagate quickly among the bacterial population, to the harm of the bacterial host. [ 13 ]


Discovered in 2008 in a strain of  Acanthamoeba castellanii  mimivirus , [ 18 ]   virophages  are an element of the virus mobilome. [ 5 ]  Virophages are viruses that  replicate  only when host cells are co-infected with  helper viruses . [ 19 ]  Following co-infection, helper viruses exploit the host cell's transcription/translation machinery to produce their own machinery; virophages replicate through the machinery of either the host cell or the viruses. [ 19 ]  The replication of virophages can negatively impact the replication of helper viruses. [ 18 ] [ 20 ]


Sputnik [ 18 ] [ 21 ]  and  mavirus [ 22 ]  are examples of virophages.


The term  modifications  in genetics refers to both naturally occurring and engineered changes in DNA.
Incidental, or natural  mutations  occur through errors during replication and repair, either spontaneously or due to environmental stressors. Intentional modifications are done in a laboratory for various purposes, developing hardier seeds and plants, and increasingly to treat human disease. The use of gene editing technology remains controversial.


Modifications  are changes in an individual's DNA due to incidental  mutation  or intentional  genetic modification  using various biotechnologies. [ 1 ]  Although confusion exists between the terms "modification" and "mutation" as they are often used interchangeably, modification differentiates itself from mutation because it acts as an umbrella term, encompassing both definitions of mutation and genetic engineering. [ 1 ]  Both of these subcategorizations result in a change affect an organism's obervable characteristics, also known as their  phenotype , caused due to alterations in an organism's genotype, or their specific alleles, resulting in altered gene expression. [ 2 ]  Although heritability plays a large role in an individual's expression, like in cases of  epigenetic  modifications, not all instances of modification are heritable. No matter the origins of such variation at the genetic level, it clearly impacts the creation and interaction of proteins, changing cell function, phenotype, and organism function. [ 3 ]


Genetic modifications can occur naturally, through aforementioned mutations in an organism's genome, or through biotechnological methods of selecting a gene of interest to manipulate in order to make something new or improve upon what already exists. [ 1 ]  This distinction between changes that occur naturally and those that are intentional is key to understanding the difference between mutation and genetic engineering. [ 1 ]


Mutation can be more accurately defined as any non-combinatorial change in phenotype that is able to be consistently inherited from parent to offspring over generations. [ 1 ]  Mutations can be attributed to many factors and come in numerous different forms, however they can mostly be attributed to mistakes that occur during  DNA replication  or exposure to external factors. [ 4 ]  As cellular processes are highly efficient, they are not perfect causing disparities between organisms of the same species. [ 4 ]  These disparities can cause many different  phenotypic effects  of all intensities, ranging from no observable impact at all to possible inviability. [ 4 ]  Due to environmental conditions such as climate, diet, oxygen levels, light cycles, and mutagens or chemicals which are strongly related to disease susceptibility, genes expression can vary. [ 5 ] [ 6 ]  The timing and duration of exposure to such elements is a critical factor as well as it can significantly impact the phenotypic response of an organism, generally increasing severity with time. [ 7 ]


Methods:


There are several methods, or forms, of  mutation  that exist including spontaneous mutation, errors during replication and repair, as well as mutation due to environmental effects. [ 8 ]  These origins of mutations can cause many different  types of mutations  which influence gene expression on both large and small scales. [ 8 ]


Genetic engineering is a type of intentional genetic modification, which uses biotechnology to alter an organism's genome. [ citation needed ]  According to  World Health Organization  (WHO), genetically modified organisms are defined as "Organisms (i.e. plants, animals or microorganisms) in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination”. [ 9 ]  This type of modification can involve insertions or deletions of DNA bases into the existing genetic code. [ 10 ]  In biotechnological methodology, a series of four steps are used in order to create a genetically modified organism (GMO). [ 11 ]


CRISPR  methods are a popularly used type of the aforementioned process of genome editing. [ 12 ]  Standing for 'Clustered Regularly Interspaced Short Palindromic Repeats', CRISPR gene editing allows scientists to manually alter gene expression, correcting errors or creating new variations. [ 12 ]  Since 2012, scientists have worked to develop this technology which has the opportunity to both cure genetic diseases and genetically modify traits to be most desirable, purposefully altering DNA with a high degree of precision. [ 13 ]


The  dandelion : Most dandelions have long stems, but an increase in potential threats in their environment have caused average dandelion stem length to decrease within certain species, allowing them to better avoid said threats. [ 14 ]  This adaptation was possible due to a mutation occurring in a shorter-stemmed individual being  selected  by environmental pressures. [ 15 ]  Because the shorter-stemmed dandelions had higher  fitness  than long-stemmed dandelions and were able to survive more often, the genetic frequency of the population was altered, genetically modified through the original occurrence of a mutation. [ 15 ]


Sickle cell disease : In a healthy individual, the  HBB  gene is responsible for encoding hemoglobin which carries oxygen throughout the body. [ 16 ]  However, when a person has this disease due to inheriting two mutated copies of the HBB gene due to a base pair point mutation, their red blood cells are shaped differently. [ 16 ]  This altered shape results in blockages of blood flow with serious health implications. [ 16 ]  On the other hand, those who inherit only one mutated copy of this gene have higher protection against  malaria . [ 17 ]


Alzheimer's disease : In a synthetic example in a laboratory, scientists isolated the  amyloid precursor protein  (APP) gene, known for using Alzheimer's in humans, and transmitted it into the nerve cells of worms. [ 10 ]  In doing this, scientists aimed to study the progression of Alzheimer's disease in this simple organism by tagging the APP protein with green fluorescent protein which allowed them to better visualize the gene as the worm aged. [ 10 ]  Using what they learned from experimentation with the simple worm and the APP gene, scientists increased their understanding of this gene's role in causing Alzheimer's disease in humans. [ 10 ]


Insulin : The first use of  genetically modified bacteria  was for the medical insulin that diabetics need to medically control their blood sugar. [ 18 ]  Through the following steps, scientists are able to genetically engineer a medical product that millions of people rely on worldwide: [ 10 ]


Fast-paced developments in the CRISPR-Cas9 gene editing technology has increased both the concerns and relevance of this ethical controversy as it has become more popularly used. [ 19 ] [ 20 ]  The scientific community recommends continued evaluation of risks and benefits of utilizing genetically modified organisms in everyday life. [ 21 ]  Genetic modifications are studied by researchers under controlled conditions after they are inserted into an organism, allowing for improved scientific understanding of the effects of certain gene modifications and certain organism responses.


In April 2015, gene editing technology was used on human embryos and debate about the ethics of such actions persisted since. [ 22 ]  Nonetheless, scientists and policymakers are in agreement that public deliberations should decide the legality of germ line genome editing. [ 23 ]  Modifying a person's non-heritable DNA with the goal of improving one's medical condition is generally accepted and has a plethora of ethical protocols monitoring such procedures. [ 19 ]  This includes modifications like organ donation, bone marrow transplants, and types of gene therapies, all of which consider cultural and religious values. [ 19 ]  On the other hand, there is contention surrounding heritable gene modification exemplified by the fact that 19 countries have outlawed this type of genetic modification. [ 19 ]  For those who believe the vitility of a human embryo is equivalent to an adult, genome editing in early development occurring at or immediately following fertilization raises moral concerns. [ 13 ]  In order to mitigate these concerns, studies using human embryos have used embryos from left over  IVF  treatments. [ 13 ]  Scientists have also suggested creating fertilized zygotes from donated sperm and eggs strictly for research purposes. [ 13 ]  However, this raises an additional ethical concern within the scientific community about the concept of a zygote being created only to be used for experimentation. [ 13 ]


Debate also surrounds genetically engineered food in terms of the controversial health and environmental effects that it may have in various time scales. [ 24 ]  Regulations have been implemented for approval of genetically modified foods to reduce some uncertainty that remains in this field. [ 25 ]  The reasons in favor of development of genetically modified foods include to meet the demands of the exponentially growing human population, to substitute for the decrease in farmable land, and to address the decrease in genetic diversity which limits possible improvement of species. [ 24 ]  Additional benefits include improved herbicide tolerance, increased pest and bacterial/fungal/viral resistance, higher stress tolerance, and increased nutrient content within the organism. [ 26 ]  The biotechnology of genetic engineering provides the opportunity to achieve global food security by addressing these problems and positively impacting the food production economy. [ 24 ]  Potential health risks are also being researched and there are requirements for the safety of genetically modified foods to be clarified before they are consumed by the public. Environmental consequences are also considered due to disruptions within the food web when these organisms are added to a previously balanced ecosystem. [ 24 ]  As genetic modification is so fast, the environment may not be able to adapt and integrate the new organism into the ecosystem or it could have unwanted effects on its surroundings. [ 26 ]  Other impacts on the environment include unnatural gene flow, modification of soil and water chemistry, and reduction of species diversity. [ 25 ]


Ethical considerations regarding gene editing are largely controversial within the scientific community due to its open ended implications for the rest of society. [ 19 ]  Although no consensus has been reached, there are plans in place to utilize the available resources to continue education, scientific research as well as research on ethical, legal, and social issues associated with genetic modification. [ 20 ]


Molecular cytogenetics  combines two disciplines,  molecular biology  and  cytogenetics , and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. [ 1 ] [ 2 ]  It includes a series of techniques referred to as  fluorescence  in situ  hybridization , or FISH, in which  DNA  probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping.  Virtual karyotypes  are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome  in silico .


Fluorescence In Situ Hybridization  maps out single copy or repetitive DNA sequences through localization labeling of specific nucleic acids. The technique utilizes different DNA probes labeled with fluorescent tags that bind to one or more specific regions of the genome. [ 3 ]  It labels all individual chromosomes at every stage of cell division to display structural and numerical abnormalities that may arise throughout the cycle. This is done with a probe that can be locus specific, centromeric, telomeric, and whole-chromosomal. This technique is typically performed on interphase cells and paraffin block tissues. FISH maps out single copy or repetitive DNA sequences through localization labeling of specific nucleic acids. The technique utilizes different DNA probes labeled with fluorescent tags that bind to one or more specific regions of the genome. Signals from the fluorescent tags can be seen with  microscopy , and mutations can be seen by comparing these signals to healthy cells. For this to work, DNA must be denatured using heat or chemicals to break the hydrogen bonds; this allows hybridization to occur once two samples are mixed. The fluorescent probes create new hydrogen bonds, thus repairing DNA with their complementary bases, which can be detected through microscopy. FISH allows one to visualize different parts of the chromosome at different stages of the cell cycle. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping.  Virtual karyotypes  are generated from microarrays made of thousands to millions of probes, and computational tools are used to recreate the genome  in silico . [ 4 ]


Comparative genomic hybridization (CGH), derived from FISH, is used to compare variations in copy number between a biological sample and a reference. CGH was originally developed to observe chromosomal aberrations in tumour cells. This method uses two genomes, a sample and a control, which are labeled fluorescently to distinguish them. [ 5 ]  In CGH, DNA is isolated from a tumour sample and biotin is attached. Another labelling protein, digoxigenin, is attached to the reference DNA sample. [ 6 ]  The labelled DNA samples are co-hybridized to probes during cell division, which is the most informative time for observing copy number variation. [ 7 ]  CGH uses creates a map that shows the relative abundance of DNA and chromosome number. By comparing the fluorescence in a sample compared to a reference, CGH can point to gains or losses of chromosomal regions. [ 6 ] [ 8 ]  CGH differs from FISH because it does not require a specific target or previous knowledge of the genetic region being analyzed. CGH can also scan an entire genome relatively quickly for various chromosome imbalances, and this is helpful in patients with underlying genetic issues and when an official diagnosis is not known. This often occurs with hematological cancers.


Array comparative genomic hybridization (aCGH) allows CGH to be performed without cell culture and isolation. Instead, it is performed on glass slides containing small DNA fragments. [ 9 ]  Removing the cell culture and isolation step dramatically simplifies and expedites the process. Using similar principles to CGH, the sample DNA is isolated and fluorescently labelled, then co-hybridized to single stranded probes to generate signals. Thousands of these signals can be detected for at once, and this process is referred to as parallel screening. [ 10 ]  Fluorescence ratios between the sample and reference signals are measured, representing the average difference between the amount of each. This will show if there is more or less sample DNA than is expected by reference.


FISH chromosome in-situ hybridization allows the study cytogenetics in pre- and postnatal samples and is also widely used in cyto genetic testing  for cancer. While cytogenetics is the study of chromosomes and their structure, cytogenetic testing involves the analysis of cells in the blood, tissue, bone marrow, or fluid to identify changes in chromosomes of an individual. This was often done through karyotyping, and is now done with FISH. This method is commonly used to detect chromosomal deletions or translocations often associated with cancer. FISH is also used for melanocytic lesions, distinguishing atypical melanocytic or malignant melanoma. [ 5 ]


Cancer cells often accumulate complex chromosomal structural changes such as loss, duplication, inversion or movement of a segment. [ 11 ]  When using FISH, any changes to a chromosome will be made visible through discrepancies between fluorescent-labelled cancer chromosomes and healthy chromosomes. [ 11 ]  The findings of these cytogenetic experiments can shed light on the genetic causes for the cancer and can locate potential therapeutic targets. [ 12 ]


Molecular cytogenetics can also be used as a diagnostic tool for  congenital syndromes  in which the underlying genetic causes of the disease are unknown. [ 13 ]  Analysis of a patient's chromosome structure can reveal causative changes. New molecular biology methods developed in the past two decades such as  next generation sequencing  and  RNA-seq  have largely replaced molecular cytogenetics in diagnostics, but recently the use of derivatives of FISH such as multicolour FISH and multicolour banding (mBAND) has been growing in medical applications. [ 14 ]


One of the current projects involving Molecular Cytogenetics involves genomic research on rare cancers, called the  Cancer Genome Characterization Initiative (CGCI) . [ 15 ]  The CGCI is a group interested in describing the genetic abnormalities of some rare cancers, by employing advanced sequencing of genomes, exomes, and transcriptomes, which may ultimately play a role in cancer pathogenesis. [ 15 ]  Currently, the CGCI has elucidated some previously undetermined genetic alterations in  medulloblastoma  and  B-cell non-Hodgkin lymphoma . The next steps for the CGCI is to identify genomic alternations in  HIV+ tumors  and in  Burkitt's Lymphoma .


Some high-throughput sequencing techniques that are used by the CGCI include:  whole genome sequencing ,  transcriptome  sequencing,  ChIP-sequencing , and  Illumina Infinum MethylationEPIC BeadCHIP . [ 16 ]