Personalized medicine  is an emerging practice in medicine that develops patient-specific treatments based on an individual's genetic profile. The treatment enables patients to experience maximized therapeutic effectiveness and minimized adverse effects. Personalized medicine has been widely accepted, and future-oriented changes in policy and infrastructure are implemented throughout the world to readily adopt into other fields.


Efforts to explore genes and heredity have been prolonged for over 100 years. From  Gregor Mendel ’s studies of  inheritance , [ 1 ]  many researchers have dedicated themselves to scientific development via new discoveries and inventions, such as the  DNA double helix  discovered by  Rosalind Franklin  and the  sanger sequencing [ 2 ]  invented by  Frederick Sanger . Recognition of chromosomal disorders like  trisomy  in 1959-1960 [ 3 ]  has led scientists to realize that genes are related to the phenotypic distinctive diseases. It motivated scientists all over to carry out several projects together to understand the human genome.


Human Genome Project  (HGP) [ 4 ]  is a research project conducted by universities and research centers throughout six countries with the primary goal of determining the complete sequence of bases of the entire human genome and identifying the complete set of human genes. This project also stored the genetic information in public databases and had a role in the improvement of tools for data analysis. A group of anonymous human volunteers recruited from diverse populations was sequenced. The establishment of HGP accelerated tracing human origins by detecting genetic mutations and constructing genetic maps. Additionally, it helped to launch other research projects around the world.


International HapMap Project [ 5 ]  is an international research project facilitating identification of genotype-phenotype correlation by  genome-wide association study  (GWAS). This research created a haplotype map of the human genome to find genetic variations that affect human disease from 11 global populations. The project focused mainly on  single nucleotide polymorphisms  (SNPs) distributed within and across populations, and designed high density SNP arrays to screen for polymorphisms, allowing researchers to search for genes involved in common human diseases.


These projects have been completed with the aid of DNA sequencing techniques which allow for the identification of genetic variations in each genome, thus personally diagnosing genetic diseases.


Personalized genomics includes the analysis of the genome of individuals. [ 2 ]  To analyze the genome, a basic knowledge of  genetics  is required. There are DNA building blocks that are called nucleotides and those nucleotides are annealed to form a strand. Two strands of DNA are  supercoiled  into double helix form and that is what people think of first when they imagine the DNA structure. These DNA are very important as they contain genetic information. The order of nucleotides determines the genomics of individuals, and individuals have different genomics . [ 2 ]  Therefore, people have different characteristics such as eye color. Not only characteristics can be revealed by DNA, but different causes of unique diseases can be identified by determining the order of nucleotides through  DNA sequencing .


To analyze personal genomics, a technique called DNA sequencing is needed and it is used to determine any disorders or polymorphisms in DNA sequences. There are two methods to conduct DNA sequencing, Whole Exome Sequencing (WES) [ 2 ]  and Whole Genome Sequencing (WGS). [ 6 ]  Formal way of sequencing, the sanger technique had some limitations that it was costly and time-consuming. The recent development of Next Generation Sequencing (NGS) [ 7 ]  dramatically remedied the shortcomings of Sanger sequencing.


NGS enabled the sequencing of large loads of DNA. [ 7 ]  There are pieces of genes that transcribe the proteins. These pieces of gene give instructions to make complementary proteins and are called exons.  Exons  are known to make up 1 percent of a human's genome. [ 2 ]  All the exons in a genome together are called the exome. Whole exome sequencing is the method to sequence all exons in genomics. As proteins produced by the exons are crucial for human metabolism and the endosomal environment, if mutations occur at the exons, it will lead to a critical genetic disease. Hence, most mutations that cause known genetic diseases arise in the exon area. WES is an efficient way to sequence possible disease-causing mutations.


WGS, on the other hand, literally stands for the method that sequences every nucleotide of an individual's DNA and can detect any variations in any location in the genome. [ 6 ]  Researchers have found that mutations at outside of exon areas also can influence gene transcription and protein production. [ 8 ]  In other words, DNA variation at the non-exon area can induce genetic disorders that WES can not detect. Hence, there are studies that conduct both WES and WGS together to increase the quality of genomic data to identify genetic disorders. [ 9 ]


By WES and WGS, various genetic variations can be observed compared to select-gene sequencing, but the significance of the information obtained from whole-scale sequencing is mostly unknown. This is because there is no 100% causal link between genetic variation and health. [ 2 ]  There are some mutations that do not affect human health or cause disease. It cannot be deduced that identified genetic change is an inducing factor of the disease of interest. Hence, a large sample size is needed to be analyzed to identify causal variants. However, the usage of next-generation sequencing has revolutionized the individualized healthcare regimens used to treat rare diseases using personalized medicine.


Genetic medicine  refers to the application of genetics to medical care in the detection and treatment of several  phenotypic  rare hereditary disorders, and this discipline incorporates an emerging area in medicine such as personalized medicine.


Personalized medicine is a type of tailored medical treatment to the individual patients’ characteristics based on their expected response or resistance to diseases. While traditional medicine follows a ‘ one-size-fits-all ’ approach [ 10 ]  that drug is designed to treat a large population of patients, personalized medicine is prescribed to the group of patients organized by molecular diagnostic and analytic techniques relating to their genetic information to minimize adverse effects and to maximize treatment effectiveness.


The necessity for personalized medicine becomes particularly evident with the reduction in the therapeutic effectiveness of existing medicine. In a BBC news interview with  Allen D. Roses , a former senior vice president at  GlaxoSmithKline [ 11 ]  claimed that 90% of drugs have a therapeutic effect on only 30-50% of patients. Patients have experienced varying success rates from drugs distributed in the market with a low efficacy rate of 50% or below. [ 11 ]  He emphasized that different genetic information contributes to variability in the response to the medication between patients. As an example of cancer cells,  tumor heterogeneity  has been found to be associated with the clinical outcome of  cancer immunotherapy , [ 12 ]  so diagnostic techniques with high accuracy must be utilized.


Accurate diagnosis of patients’ circumstances must be performed to discover  biomarkers , which are biological indicators of medical state which can be measured objectively and accurately. A prognostic signature developed by biomarker discovery is predictive of disease progression and patients’ response to drug intervention. Consequently, it can be used in the development of new pharmaceuticals and in the selection of eligible patients who are expected to be treated with certain medications. For example, single molecular array technology [ 13 ]  has detected changes in prostate-specific  antigen  levels between each  prostate cancer  cell. Moreover, fiber microarrays [ 13 ]  have been served as a multiplexed diagnostics platform for identifying potential biomarkers of  cystic fibrosis , including  interleukin-8 , [ 14 ]   vascular endothelial growth factor , [ 15 ]   epidermal growth factor , [ 13 ]  interferon-gamma inducible protein, [ 13 ]  and  matrix metallopeptidase-9 . [ 13 ]  These biomarker signatures enabled patients to be differentiated into subgroups depending on their disease severity.


The integration of diagnostic techniques and genomic sequencing has played a vital role in allowing for the implementation of personalized medicine in the market. For instance,  warfarin [ 16 ]  may lead to acute  cardiac failure ,  hemorrhage ,  necrosis , and  osteoporosis  depending on the genetic variations in the  CYP2C9 , [ 16 ]  and  VKORC1  gene. [ 16 ]  Since the prescription dose for warfarin varies by approximately 10-fold based on the genetic variation of each patient,  U.S. Food and Drug Administration  (FDA) [ 16 ]  recommends that warfarin dosing should be prescribed after conducting genetic testing. In addition,  gefitinib , an inhibitor of  epidermal growth factor receptor  (EGFR) - tyrosine kinase , has therapeutic efficacy on only  breast cancer [ 17 ]  or  non-small cell lung cancer . [ 18 ]  Genetic variations in the EGFR gene have been reported to be associated with the sensitivity of patients with lung cancer to gefitinib. [ 19 ]  Patients with breast cancer expressing activated  HER2/neu  have benefited from therapeutic effects of gefitinib. [ 20 ]  FDA-approved [ 21 ]  gefitinib should be given for the treatment of patients with breast or non-small cell lung cancer.


The price of DNA sequencing was very expensive until recently. WGS cost about $20,000 during the 2010s. [ 22 ]  WGS and WES are relatively recent technologies in clinical practice. Current data of health economic evidence to encourage use of WES and WGS in clinical research is restricted. Hence, there are not enough studies that carefully evaluate and indicate the cost-effectiveness of these technologies and components that are included in cost estimates. However, the development of  Artificial Intelligence  (AI) and the knowledge of big data and machine learning, [ 13 ]  enabled researchers to analyze massive loads of DNA sequencing faster and efficiently. Now the price of WGS fell to its lowest, $1,500. Single tests for WES range from $555 to $5,169 and for WGS from $1,906 to $24,810. [ 23 ]  It is decreasing with the development of technologies. Currently, due to the notable decrease in the cost, countries apart from the developed countries, are planning to do WGS of the unique patients or by conducting joint study to get a foundation to form a big data of population genetics.


The importance of engineering and computation has been brought to the attention of clinics and the marketplace in compliance with their incorporation into the medical field and growing public demand for individualized health care services. Computer-based analytical platforms using AI, [ 13 ]  and  Machine Learning [ 13 ]  are practicable for clinical trial use. They were able to determine patient-specific optimal therapies based on subpopulation-specific stratifications classified by genome sequencing. According to the study guided by the AI platform, [ 24 ]  liver transplant immunosuppressant prescriptions were successfully recommended depending on the patient's genetic information, and it confirmed that there was less inter-patients variance in AI-derived drug recommendation compared to control cohort patients. Computational analytical platforms predicted patients’ response towards drugs and boosted the process of recreating medical drugs and device validation. [ 13 ]  The integration of computer programming and personalized medicine enables a large number of patients to receive personal care in a short period of time with greater efficiency.


The medical community follows an increasing trend of translating traditional medicine into personalized medicine, and long-held regulatory policies are obstacles to be resolved for its public usage. The United States took action via the enactment of the 21st Century Cures Act in 2016. [ 13 ]  This enforcement provided financial aid to  U.S. Food and Drug Administration  (FDA), [ 13 ]  allowing to accelerate the emergence of newly personalized products such as cell therapy and medical devices into the marketplace. Also,  National Institutes of Health  (NIH) benefited from the resource allocation. Many countries have introduced legislation for the improvement of personalized medicine. In Singapore, AI Singapore, a national AI initiative supported by National Research Foundation, [ 13 ]  was able to enhance national AI capabilities in drug development and patient-centered care. In addition, the Innovative Medicines Initiative (IMI) [ 13 ]  was established between the European Union and pharmaceutical companies with the purpose of developing medical devices, drugs, and vaccines to tackle major challenges that will imperil the EU.


Whole genome sequencing  ( WGS ) is the process of determining the entirety, or nearly the entirety, of the  DNA  sequence of an organism's  genome  at a single time. [ 2 ]  This entails sequencing all of an organism's  chromosomal  DNA as well as DNA contained in the  mitochondria  and, for plants, in the  chloroplast .


Whole genome sequencing has largely been used as a research tool, but was being introduced to clinics in 2014. [ 3 ] [ 4 ] [ 5 ]  In the future of  personalized medicine , whole genome sequence data may be an important tool to guide therapeutic intervention. [ 6 ]  The tool of  gene sequencing  at  SNP  level is also used to pinpoint functional variants from  association studies  and improve the knowledge available to researchers interested in  evolutionary biology , and hence may lay the foundation for predicting disease susceptibility and drug response.


Whole genome sequencing should not be confused with  DNA profiling , which only determines the likelihood that genetic material came from a particular individual or group, and does not contain additional information on genetic relationships, origin or susceptibility to specific diseases. [ 7 ]  In addition, whole genome sequencing should not be confused with methods that sequence specific subsets of the genome – such methods include  whole exome sequencing  (1–2% of the genome) or  SNP genotyping  (< 0.1% of the genome).


The DNA sequencing methods used in the 1970s and 1980s were manual; for example,  Maxam–Gilbert sequencing  and  Sanger sequencing . Several whole bacteriophage and animal viral genomes were sequenced by these techniques, but the shift to more rapid, automated sequencing methods in the 1990s facilitated the sequencing of the larger bacterial and eukaryotic genomes. [ 9 ]


The first virus to have its complete genome sequenced was the  Bacteriophage MS2  by 1976. [ 10 ]  In 1992, yeast chromosome III was the first chromosome of any organism to be fully sequenced. [ 11 ]  The first organism whose entire genome was fully sequenced was  Haemophilus influenzae  in 1995. [ 12 ]  After it, the genomes of other bacteria and some  archaea  were first sequenced, largely due to their small genome size.  H. influenzae  has a genome of 1,830,140 base pairs of DNA. [ 12 ]  In contrast,  eukaryotes , both  unicellular  and  multicellular  such as  Amoeba dubia  and humans ( Homo sapiens ) respectively, have much larger genomes (see  C-value paradox ). [ 13 ]   Amoeba dubia  has a genome of 700 billion  nucleotide  pairs spread across thousands of  chromosomes . [ 14 ]  Humans contain fewer  nucleotide  pairs (about 3.2 billion in each  germ cell  – note the exact size of the human genome is still being revised) than  A. dubia,  however, their genome size far outweighs the genome size of individual bacteria. [ 15 ]


The first bacterial and archaeal genomes, including that of  H. influenzae , were sequenced by  Shotgun sequencing . [ 12 ]  In 1996, the first eukaryotic genome ( Saccharomyces cerevisiae ) was sequenced.  S. cerevisiae , a  model organism  in  biology  has a genome of only around 12 million  nucleotide  pairs, [ 16 ]  and was the first  unicellular  eukaryote to have its whole genome sequenced. The first  multicellular  eukaryote, and  animal , to have its whole genome sequenced was the  nematode  worm:  Caenorhabditis elegans  in 1998. [ 17 ]  Eukaryotic genomes are sequenced by several methods including Shotgun sequencing of short DNA fragments and sequencing of larger DNA clones from  DNA libraries  such as  bacterial artificial chromosomes  (BACs) and  yeast artificial chromosomes  (YACs). [ 18 ]


In 1999, the entire DNA sequence of human  chromosome 22 , the second shortest human  autosome , was published. [ 19 ]  By the year 2000, the second animal and second  invertebrate  (yet first  insect ) genome was sequenced – that of the fruit fly  Drosophila melanogaster  – a popular choice of model organism in experimental research. [ 20 ]  The first  plant  genome – that of the model organism  Arabidopsis thaliana  – was also fully sequenced by 2000. [ 21 ]  By 2001, a draft of the entire human genome sequence was published. [ 22 ]  The genome of the laboratory mouse  Mus musculus  was completed in 2002. [ 23 ]


In 2004, the  Human Genome Project  published an incomplete version of the human genome. [ 24 ]  In 2008, a group from Leiden, the Netherlands, reported the sequencing of the first female human genome ( Marjolein Kriek ).


Currently  thousands of genomes have been wholly or partially sequenced .


Almost any biological sample containing a full copy of the DNA—even a very small amount of DNA or  ancient DNA —can provide the genetic material necessary for full genome sequencing. Such samples may include  saliva ,  epithelial cells ,  bone marrow ,  hair  (as long as the hair contains a  hair follicle ),  seeds , plant leaves, or anything else that has DNA-containing cells.


The genome sequence of a single cell selected from a mixed population of cells can be determined using techniques of  single cell genome sequencing . This has important advantages in environmental microbiology in cases where a single cell of a particular microorganism species can be isolated from a mixed population by microscopy on the basis of its morphological or other distinguishing characteristics. In such cases the normally necessary steps of isolation and growth of the organism in culture may be omitted, thus allowing the sequencing of a much greater spectrum of organism genomes. [ 25 ]


Single cell genome sequencing is being tested as a method of  preimplantation genetic diagnosis , wherein a cell from the embryo created by  in vitro fertilization  is taken and analyzed before  embryo transfer  into the uterus. [ 26 ]  After implantation,  cell-free fetal DNA  can be taken by simple  venipuncture  from the mother and used for whole genome sequencing of the fetus. [ 27 ]


Sequencing of nearly an entire human genome was first accomplished in 2000 partly through the use of  shotgun sequencing  technology. While full genome shotgun sequencing for small (4000–7000  base pair ) genomes was already in use in 1979, [ 28 ]  broader application benefited from pairwise end sequencing, known colloquially as  double-barrel shotgun sequencing . As sequencing projects began to take on longer and more complicated genomes, multiple groups began to realize that useful information could be obtained by sequencing both ends of a fragment of DNA. Although sequencing both ends of the same fragment and keeping track of the paired data was more cumbersome than sequencing a single end of two distinct fragments, the knowledge that the two sequences were oriented in opposite directions and were about the length of a fragment apart from each other was valuable in reconstructing the sequence of the original target fragment.


The first published description of the use of paired ends was in 1990 as part of the sequencing of the human  HPRT  locus, [ 29 ]  although the use of paired ends was limited to closing gaps after the application of a traditional shotgun sequencing approach. The first theoretical description of a pure pairwise end sequencing strategy, assuming fragments of constant length, was in 1991. [ 30 ]  In 1995, the innovation of using fragments of varying sizes was introduced, [ 31 ]  and demonstrated that a pure pairwise end-sequencing strategy would be possible on large targets. The strategy was subsequently adopted by  The Institute for Genomic Research  (TIGR) to sequence the entire genome of the bacterium  Haemophilus influenzae  in 1995, [ 32 ]  and then by  Celera Genomics  to sequence the entire fruit fly genome in 2000, [ 33 ]  and subsequently the entire human genome.  Applied Biosystems , now called  Life Technologies , manufactured the automated capillary sequencers utilized by both Celera Genomics and The Human Genome Project.


While capillary sequencing was the first approach to successfully sequence a nearly full human genome, it is still too expensive and takes too long for commercial purposes. Since 2005, capillary sequencing has been progressively displaced by  high-throughput  (formerly "next-generation") sequencing technologies such as  Illumina dye sequencing ,  pyrosequencing , and  SMRT sequencing . [ 34 ]  All of these technologies continue to employ the basic shotgun strategy, namely, parallelization and template generation via genome fragmentation.


Other technologies have emerged, including  Nanopore technology . Though the sequencing accuracy of Nanopore technology is lower than those above, its read length is on average much longer. [ 35 ]  This generation of long reads is valuable especially in  de novo  whole-genome sequencing applications. [ 36 ]


In principle, full genome sequencing can provide the raw  nucleotide  sequence of an individual organism's DNA at a single point in time. However, further analysis must be performed to provide the biological or medical meaning of this sequence, such as how this knowledge can be used to help prevent disease. Methods for analyzing sequencing data are being developed and refined.


Because sequencing generates a lot of data (for example, there are approximately six billion  base pairs  in each human diploid genome), its output is stored electronically and requires a large amount of computing power and storage capacity.


While analysis of WGS data can be slow, it is possible to speed up this step by using dedicated hardware. [ 37 ]


A number of public and private companies are competing to develop a full genome sequencing platform that is commercially robust for both research and clinical use, [ 38 ]  including Illumina, [ 39 ]   Knome , [ 40 ]   Sequenom , [ 41 ]
 454 Life Sciences , [ 42 ]  Pacific Biosciences, [ 43 ]   Complete Genomics , [ 44 ]
 Helicos Biosciences , [ 45 ]   GE Global Research  ( General Electric ),  Affymetrix ,  IBM , Intelligent Bio-Systems, [ 46 ]  Life Technologies, Oxford Nanopore Technologies, [ 47 ]  and the  Beijing Genomics Institute . [ 48 ] [ 49 ] [ 50 ]  These companies are heavily financed and backed by  venture capitalists ,  hedge funds , and  investment banks . [ 51 ] [ 52 ]


A commonly-referenced commercial target for sequencing cost until the late 2010s was  $1,000   USD, however, the private companies are working to reach a new target of only $100. [ 53 ]


In October 2006, the  X Prize Foundation , working in collaboration with the J. Craig Venter Science Foundation, established the  Archon X Prize  for Genomics, [ 54 ]  intending to award $10 million to "the first team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 1,000,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $1,000 per genome". [ 55 ]  The  Archon X Prize  for Genomics was cancelled in 2013, before its official start date. [ 56 ] [ 57 ]


In 2007,  Applied Biosystems  started selling a new type of sequencer called SOLiD System. [ 58 ]  The technology allowed users to sequence 60 gigabases per run. [ 59 ]


In June 2009, Illumina announced that they were launching their own Personal Full Genome Sequencing Service at a  depth  of 30× for $48,000 per genome. [ 60 ] [ 61 ]  In August, the founder of Helicos Biosciences,  Stephen Quake , stated that using the company's Single Molecule Sequencer he sequenced his own full genome for less than $50,000. [ 62 ]  In November, Complete Genomics published a peer-reviewed paper in  Science  demonstrating its ability to sequence a complete human genome for $1,700. [ 63 ] [ 64 ]


In May 2011, Illumina lowered its Full Genome Sequencing service to $5,000 per human genome, or $4,000 if ordering 50 or more. [ 65 ]
Helicos Biosciences, Pacific Biosciences, Complete Genomics, Illumina, Sequenom, ION Torrent Systems, Halcyon Molecular, NABsys, IBM, and GE Global appear to all be going head to head in the race to commercialize full genome sequencing. [ 34 ] [ 66 ]


With sequencing costs declining, a number of companies began claiming that their equipment would soon achieve the $1,000 genome: these companies included  Life Technologies  in January 2012, [ 67 ]   Oxford Nanopore Technologies  in February 2012, [ 68 ]  and  Illumina  in February 2014. [ 69 ] [ 70 ]  In 2015, the  NHGRI  estimated the cost of obtaining a whole-genome sequence at around $1,500. [ 71 ]  In 2016,  Veritas Genetics  began selling whole genome sequencing, including a report as to some of the information in the sequencing for $999. [ 72 ]  In summer 2019, Veritas Genetics cut the cost for WGS to $599. [ 73 ]  In 2017, BGI began offering WGS for $600. [ 74 ]


However, in 2015, some noted that effective use of whole gene sequencing can cost considerably more than $1000. [ 75 ]  Also, reportedly there remain parts of the human genome that have not been fully sequenced by 2017. [ 76 ] [ 77 ]


Full genome sequencing provides information on a genome that is orders of magnitude larger than by  DNA arrays , the previous leader in genotyping technology.


For humans, DNA arrays currently provide genotypic information on up to one million genetic variants, [ 78 ] [ 79 ] [ 80 ]  while full genome sequencing will provide information on all six billion bases in the human genome, or 3,000 times more data. Because of this, full genome sequencing is considered a  disruptive innovation  to the DNA array markets as the accuracy of both range from 99.98% to 99.999% (in non-repetitive DNA regions) and their consumables cost of $5000 per 6 billion base pairs is competitive (for some applications) with DNA arrays ($500 per 1 million basepairs). [ 42 ]


Whole genome sequencing has established the  mutation  frequency for whole human genomes. The mutation frequency in the whole genome between generations for humans (parent to child) is about 70 new mutations per generation. [ 81 ] [ 82 ]  An even lower level of variation was found comparing whole genome sequencing in blood cells for a pair of monozygotic (identical twins) 100-year-old centenarians. [ 83 ]  Only 8 somatic differences were found, though somatic variation occurring in less than 20% of blood cells would be undetected.


In the specifically protein coding regions of the human genome, it is estimated that there are about 0.35 mutations that would change the protein sequence between parent/child generations (less than one mutated protein per generation). [ 84 ]


In cancer, mutation frequencies are much higher, due to  genome instability . This frequency can further depend on patient age, exposure to DNA damaging agents (such as UV-irradiation or components of tobacco smoke) and the activity/inactivity of DNA repair mechanisms. [ 85 ]  Furthermore, mutation frequency can vary between cancer types: in germline cells, mutation rates occur at approximately 0.023 mutations per megabase, but this number is much higher in breast cancer (1.18-1.66 somatic mutations per Mb), in lung cancer (17.7) or in melanomas (≈33). [ 86 ]   Since the haploid human genome consists of approximately 3,200 megabases, [ 87 ]  this translates into about 74 mutations (mostly in  noncoding  regions) in germline DNA per generation, but 3,776-5,312 somatic mutations per haploid genome in breast cancer, 56,640 in lung cancer and 105,600 in melanomas.


The distribution of somatic mutations across the human genome is very uneven, [ 88 ]  such that the gene-rich, early-replicating regions receive fewer mutations than gene-poor, late-replicating heterochromatin, likely due to differential DNA repair activity. [ 89 ]  In particular, the  histone modification  H3K9me3 is associated with high, [ 90 ]  and  H3K36me3  with low mutation frequencies. [ 91 ]


In research, whole-genome sequencing can be used in a Genome-Wide Association Study (GWAS) – a project aiming to determine the genetic variant or variants associated with a disease or some other phenotype. [ 92 ]


In 2009,  Illumina  released its first whole genome sequencers that were approved for clinical as opposed to research-only use and doctors at  academic medical centers  began quietly using them to try to diagnose what was wrong with people whom standard approaches had failed to help. [ 93 ]  In 2009, a team from Stanford led by  Euan Ashley  performed clinical interpretation of a full human genome, that of bioengineer Stephen Quake. [ 94 ]  In 2010, Ashley's team reported whole genome molecular autopsy [ 95 ]  and in 2011, extended the interpretation framework to a fully sequenced family, the West family, who were the first family to be sequenced on the Illumina platform. [ 96 ]  The price to sequence a genome at that time was $19,500   USD, which was billed to the patient but usually paid for out of a research grant; one person at that time had applied for reimbursement from their insurance company. [ 93 ]   For example, one child had needed around 100 surgeries by the time he was three years old, and his doctor turned to whole genome sequencing to determine the problem; it took a team of around 30 people that included 12  bioinformatics  experts, three sequencing technicians, five physicians, two genetic counsellors and two ethicists to identify a rare mutation in the  XIAP  that was causing widespread problems. [ 93 ] [ 97 ] [ 98 ]


Due to recent cost reductions (see above) whole genome sequencing has become a realistic application in DNA diagnostics. In 2013, the 3Gb-TEST consortium obtained funding from the European Union to prepare the health care system for these innovations in DNA diagnostics. [ 99 ] [ 100 ]   Quality assessment  schemes,  Health technology assessment  and  guidelines  have to be in place. The 3Gb-TEST consortium has identified the analysis and interpretation of sequence data as the most complicated step in the diagnostic process. [ 101 ]  At the Consortium meeting in Athens in September 2014, the Consortium coined the word  genotranslation  for this crucial step. This step leads to a so-called  genoreport . Guidelines are needed to determine the required content of these reports. [ citation needed ]


Genomes2People (G2P), an initiative of  Brigham and Women's Hospital  and  Harvard Medical School  was created in 2011 to examine the integration of genomic sequencing into clinical care of adults and children. [ 102 ]  G2P's director,  Robert C. Green , had previously led the REVEAL study — Risk EValuation and Education for Alzheimer's Disease – a series of clinical trials exploring patient reactions to the knowledge of their genetic risk for Alzheimer's. [ 103 ] [ 104 ]


In 2018, researchers at  Rady Children's Hospital  Institute for Genomic Medicine in  San Diego  determined that rapid whole-genome sequencing (rWGS) could diagnose genetic disorders in time to change acute medical or surgical management (clinical utility) and improve outcomes in acutely ill infants. In a retrospective cohort study of acutely ill inpatient infants in a regional children's hospital from July 2016-March 2017, forty-two families received rWGS for etiologic diagnosis of genetic disorders. The diagnostic sensitivity of rWGS was 43% (eighteen of 42 infants) and 10% (four of 42 infants) for standard genetic tests (P = .0005). The rate of clinical utility of rWGS (31%, thirteen of 42 infants) was significantly greater than for standard genetic tests (2%, one of 42; P = .0015). Eleven (26%) infants with diagnostic rWGS avoided morbidity, one had a 43% reduction in likelihood of mortality, and one started palliative care. In six of the eleven infants, the changes in management reduced inpatient cost by $800,000-$2,000,000. The findings replicated a prior study of the clinical utility of rWGS in acutely ill inpatient infants, and demonstrated improved outcomes, net healthcare savings and consideration as a first tier test in this setting. [ 105 ]


A 2018 review of 36 publications found the cost for whole genome sequencing to range from $1,906   USD to $24,810   USD and have a wide variance in diagnostic yield from 17% to 73% depending on patient groups. [ 106 ]


Whole genome sequencing studies enable the assessment of associations between complex traits and both coding and noncoding  rare variants  ( minor allele frequency  (MAF) < 1%) across the genome. Single-variant analyses typically have low power to identify associations with rare variants, and variant set tests have been proposed to jointly test the effects of given sets of multiple rare variants. [ 107 ]   SNP annotations  help to prioritize rare functional variants, and incorporating these annotations can effectively boost the power of genetic association of rare variants analysis of whole genome sequencing studies. [ 108 ]  Some tools have been specifically developed to provide all-in-one rare variant association analysis for whole-genome sequencing data, including integration of genotype data and their functional annotations, association analysis, result summary and visualization. [ 109 ] [ 110 ]


Meta-analysis of whole genome sequencing studies provides an attractive solution to the problem of collecting large sample sizes for discovering rare variants associated with complex phenotypes. Some methods have been developed to enable functionally informed rare variant association analysis in biobank-scale cohorts using efficient approaches for summary statistic storage. [ 111 ]


In this field, whole genome sequencing represents a great set of improvements and challenges to be faced by the scientific community, as it makes it possible to analyze, quantify and characterize  circulating tumor DNA  (ctDNA) in the bloodstream. This serves as a basis for early cancer diagnosis, treatment selection and  relapse  monitoring, as well as for determining the mechanisms of resistance,  metastasis  and  phylogenetic  patterns in the evolution of cancer. It can also help in the selection of individualized treatments for patients suffering from this pathology and observe how existing drugs are working during the progression of treatment. Deep whole genome sequencing involves a subclonal reconstruction based on ctDNA in plasma that allows for complete  epigenomic  and  genomic  profiling, showing the expression of circulating tumor DNA in each case.
 [ 112 ]


In 2013, Green and a team of researchers launched the BabySeq Project to study the ethical and medical consequences of sequencing a newborn's DNA. [ 113 ] [ 114 ]
As of 2015, whole genome and exome sequencing as a  newborn screening  tool were deliberated [ 115 ]  and in 2021, further discussed. [ 116 ]


In 2021, the NIH funded BabySeq2, an implementation study that expanded the BabySeq project, enrolling 500 infants from diverse families and track the effects of their genomic sequencing on their pediatric care. [ 117 ]


In 2023, the Lancet opined that in the UK "focusing on improving screening by upgrading targeted gene panels might be more sensible in the short term. Whole genome sequencing in the long term deserves thorough examination and universal caution." [ 118 ]


The introduction of whole genome sequencing may have ethical implications. [ 119 ]  On one hand, genetic testing can potentially diagnose preventable diseases, both in the individual undergoing genetic testing and in their relatives. [ 119 ]  On the other hand, genetic testing has potential downsides such as  genetic discrimination , loss of anonymity, and psychological impacts such as discovery of  non-paternity . [ 120 ]


Some ethicists insist that the privacy of individuals undergoing genetic testing must be protected, [ 119 ]  and is of particular concern when  minors  undergo genetic testing. [ 121 ]  Illumina's CEO, Jay Flatley, wrongly claimed in February 2009 that "by 2019 it will have become routine to map infants' genes when they are born". [ 122 ]  This potential use of genome sequencing is highly controversial, as it runs counter to established  ethical   norms  for predictive  genetic testing  of asymptomatic minors that have been well established in the fields of  medical genetics  and  genetic counseling . [ 123 ] [ 124 ] [ 125 ] [ 126 ]  The traditional guidelines for genetic testing have been developed over the course of several decades since it first became possible to test for genetic markers associated with disease, prior to the advent of cost-effective, comprehensive genetic screening. [ citation needed ]


When an individual undergoes whole genome sequencing, they reveal information about not only their own DNA sequences, but also about probable DNA sequences of their close genetic relatives. [ 119 ]  This information can further reveal useful predictive information about relatives' present and future health risks. [ 127 ]  Hence, there are important questions about what obligations, if any, are owed to the family members of the individuals who are undergoing genetic testing. In Western/European society, tested individuals are usually encouraged to share important information on any genetic diagnoses with their close relatives, since the importance of the genetic diagnosis for offspring and other close relatives is usually one of the reasons for seeking a genetic testing in the first place. [ 119 ]  Nevertheless, a major ethical dilemma can develop when the patients refuse to share information on a diagnosis that is made for serious genetic disorder that is highly preventable and where there is a high risk to relatives carrying the same disease mutation. Under such circumstances, the clinician may suspect that the relatives would rather know of the diagnosis and hence the clinician can face a conflict of interest with respect to patient-doctor confidentiality. [ 119 ]


Privacy concerns can also arise when whole genome sequencing is used in scientific research studies. Researchers often need to put information on patient's genotypes and phenotypes into public scientific databases, such as locus specific databases. [ 119 ]  Although only anonymous patient data are submitted to locus specific databases, patients might still be identifiable by their relatives in the case of finding a rare disease or a rare missense mutation. [ 119 ]  Public discussion around the introduction of advanced forensic techniques (such as advanced familial searching using public DNA ancestry websites and DNA phenotyping approaches) has been limited, disjointed, and unfocused. As forensic genetics and medical genetics converge toward genome sequencing, issues surrounding genetic data become increasingly connected, and additional legal protections may need to be established. [ 128 ]


The first nearly complete human genomes sequenced were two Americans of predominantly  Northwestern European  ancestry in 2007 ( J. Craig Venter  at 7.5-fold  coverage , [ 129 ] [ 130 ] [ 131 ]  and  James Watson  at 7.4-fold). [ 132 ] [ 133 ] [ 134 ]  This was followed in 2008 by sequencing of an anonymous  Han Chinese  man (at 36-fold), [ 135 ]  a  Yoruban  man from  Nigeria  (at 30-fold), [ 136 ]  a female clinical geneticist ( Marjolein Kriek ) from the Netherlands (at 7 to 8-fold), and a female  leukemia  patient in her mid-50s (at 33 and 14-fold coverage for tumor and normal tissues). [ 137 ]   Steve Jobs  was among the first 20 people to have their whole genome sequenced, reportedly for the cost of $100,000. [ 138 ]  As of June 2012 [update] , there were 69 nearly complete human genomes publicly available. [ 139 ]  In November 2013, a Spanish family made their personal genomics data publicly available under a  Creative Commons public domain license . The work was led by  Manuel Corpas  and the data obtained by  direct-to-consumer genetic testing  with  23andMe  and the  Beijing Genomics Institute . This is believed to be the first such  Public Genomics  dataset for a whole family. [ 140 ]


According to  Science , the major databases of whole genomes are: [ 141 ]


In terms of  genomic coverage  and accuracy, whole genome sequencing can broadly be classified into either of the following: [ 144 ]


Producing a truly high-quality  finished  sequence by this definition is very expensive. Thus, most human "whole genome sequencing" results are  draft sequences  (sometimes above and sometimes below the accuracy defined above). [ 144 ]


Acridine Orange Base
Acridine Orange NO
Basic Orange 14
Euchrysine
Rhoduline Orange
Rhoduline Orange N
Rhoduline Orange NO
Solvent Orange 15


Acridine orange  is an  organic compound  that serves as a  nucleic acid -selective  fluorescent   dye  with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows it to interact with  DNA  by  intercalation , or  RNA  via  electrostatic attractions . When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein.


Acridine orange is used in  epifluorescence microscopy  and  flow cytometry . The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows it to differentiate between various types of cells (i.e., bacterial cells and white blood cells).


Acridine orange and  fluorescein  have a maximum excitation at 502 nm  and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as  lysosomes  and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of  phagocytosis  of apoptotic cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain. [ 1 ]


When the  pH  of the environment is 3.5, acridine orange becomes excited by blue light (460 nm). When acridine orange is excited by blue light, the fluorescent dye can differentially stain human cells green and prokaryotic cells orange (600 nm), allowing for rapid detection with a fluorescent microscope. The differential staining capability of acridine orange provides quick scanning of specimen smears at lower magnifications of 400x compared to  Gram stains  that operate at 1000x magnification. The differentiation of cells is aided by a dark background that allows colored organisms to be easily detected. The sharp contrast provides a mechanism for counting the number of organisms present in a sample. When acridine orange binds to DNA, the dye exhibits a maximum excitation at 502 nm producing a maximum emission of 525 nm. When bound to RNA, acridine orange displays a maximum emission value of 650 nm and a maximum excitation value of 460 nm. The maximum excitation and emission value that occur when acridine orange is bound to RNA are the result of electrostatic interactions and the intercalation between the acridine molecule and nucleic acid-base pairs present within RNA and DNA. [ 2 ]


Acridine dyes are prepared via the condensation of  1,3-diaminobenzene  with suitable benzaldehydes. Acridine orange is derived from dimethylamino benzaldehyde  and  N , N -dimethyl-1,3-diaminobenzene. [ 3 ]  It may also be prepared by the  Eschweiler–Clarke reaction  of 3,6-Acridinediamine.


Acridine orange is derived from the organic molecule acridine, which was first discovered by Carl Grabe and Heinrich Caro, who isolated acridine by boiling coal in Germany during the late nineteenth century. Acridine has antimicrobial factors useful in drug-resistant bacteria and isolating bacteria in various environments. [ 4 ]  Acridine orange in the mid-twentieth century was used to examine the microbial content found in soil and direct counts of aquatic bacteria. Additionally, the method of acridine orange direct count (AODC) proved useful in the enumeration of bacteria found within landfills. Direct epifluorescent filter technique (DEFT) using acridine orange is a method known for examining the microbial content within food and water. The use of acridine orange in clinical applications has become widely accepted, mainly focusing on highlighting bacteria in blood cultures. Past and present studies comparing acridine orange staining with blind subcultures for the detection of positive blood cultures showed that the acridine orange is a simple, inexpensive, rapid staining procedure that appears to be more sensitive than the Gram stain for detecting microorganisms in cerebrospinal fluid and other clinical and non-clinical materials. [ 3 ]


Acridine orange has been widely accepted and used in many different areas, such as epifluorescence microscopy, and the assessment of  sperm   chromatin  quality. Acridine orange is useful in the rapid screening of ordinarily sterile specimens. When acridine orange is used with flow cytometry, the differential stain is used to measure  DNA denaturation [ 5 ]  and the cellular content of DNA versus RNA [ 6 ]  in individual cells, or detect  DNA damage   in infertile sperm cells. [ 7 ]  Acridine orange is recommended for the use of fluorescent microscopic detection of microorganisms in smears prepared from clinical and non-clinical materials. Acridine orange staining has to be performed at an acidic  pH  to obtain the differential staining, which allows bacterial cells to stain orange and tissue components to stain yellow or green. [ 8 ]


Acridine orange is recorded as being used as a curing agent to cure  selectable marker  in antibiotic resistant organisms present in a sample. When isolates are subjected to curing in the presence of acridine orange, a substantial number were recorded to have been cured of at least one resistant marker. [ citation needed ]  Acridine orange is also used to stain acidic  vacuoles  ( lysosomes ,  endosomes , and  autophagosomes ), RNA, and DNA in living cells. This method is a cheap and easy way to study lysosomal  vacuolation ,  autophagy , and  apoptosis . The emission color of acridine orange changes from yellow, to orange, to red as the pH drops in an acidic vacuole of the living cell. Under specific conditions of ionic strength and concentration, acridine orange emits red fluorescence when it binds to RNA by  stacking  interactions, and green fluorescence when it binds to DNA by  intercalation . Depending on acridine orange concentration,  nuclei  may emit yellowish-green fluorescence in untreated cells, and green fluorescence when  RNA synthesis  is inhibited by compounds such as  chloroquine . [ 9 ]  Acridine orange can be used in conjunction with  ethidium bromide  or  propidium iodide  to differentiate between viable,  apoptotic , and  necrotic  cells. Additionally, acridine orange may be used on blood samples causing  bacterial DNA  to fluoresce, aiding in the clinical diagnosis of bacterial infections, such as meningitis. [ 3 ]


In  molecular biology , an  amplicon  is a piece of  DNA  or  RNA  that is the source and/or product of  amplification  or  replication  events. It can be formed artificially, using various methods including  polymerase chain reactions  (PCR) or  ligase chain reactions  (LCR), or naturally through  gene duplication . In this context,  amplification  refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction,  amplicon  is used interchangeably with common laboratory terms, such as "PCR product."


Artificial amplification is used in  research , [ 1 ]   forensics , [ 2 ]  and  medicine [ 1 ]  for purposes that include detection and quantification of  infectious agents , [ 3 ]  identification of human remains, [ 4 ]  and extracting  genotypes  from human hair. [ 2 ]


Natural  gene duplication  plays a major role in  evolution . It is also implicated in several forms of human cancer including  primary mediastinal B cell lymphoma  and  Hodgkin's lymphoma . [ 5 ]  In this context the term  amplicon  can refer both to a section of  chromosomal  DNA that has been excised, amplified, and reinserted elsewhere in the  genome , and to a fragment of extrachromosomal DNA known as a  double minute , each of which can be composed of one or more  genes . Amplification of the genes encoded by these amplicons generally increases  transcription  of those genes and ultimately the volume of associated  proteins . [ 6 ]


Amplicons in general are  direct repeat  (head-to-tail) or  inverted repeat  (head-to-head or tail-to-tail) genetic sequences, and can be either linear or circular in structure. [ 7 ]  Circular amplicons consist of imperfect inverted duplications  annealed  into a circle [ 8 ]  and are thought to arise from precursor linear amplicons. [ 9 ]


During artificial amplification, amplicon length is dictated by the experimental goals. [ 10 ]


Analysis of amplicons has been made possible by the development of amplification methods such as  PCR , and increasingly by cheaper and more  high-throughput  technologies for  DNA sequencing  or  next-generation sequencing , such as  ion semiconductor sequencing , popularly referred to as the brand of the developer, Ion Torrent. [ 11 ]


DNA sequencing technologies such as next-generation sequencing have made it possible to study amplicons in  genome biology  and  genetics , including  cancer genetics  research, [ 12 ]   phylogenetic  research, and  human genetics . [ 13 ]  For example, using the  16S rRNA  gene, which is part of every bacterial and archaeal genome and is highly conserved, bacteria can be taxonomically classified by comparison of the amplicon sequence to known sequences. This works similarly in the fungal domain with the  18S rRNA  gene as well as the  ITS1  non-coding region. [ 14 ]


Irrespective of the approach used to amplify the amplicons, some technique must be used to quantitate the amplified product. [ 15 ]  Generally, these techniques incorporate a capture step and a detection step, although how these steps are incorporated depends on the individual  assay .


Examples include the Amplicor HIV-1 Monitor Assay ( RT-PCR ), which has the capacity to recognize  HIV  in  plasma ; the HIV-1 QT ( NASBA ), which is used to measure plasma  viral load  by amplifying a segment of the HIV RNA; and  transcription mediated amplification , which employs a hybridization protection assay to distinguish  Chlamydia trachomatis  infections. [ 15 ]  Various detection and capture steps are involved in each approach to assess the amplification product, or amplicon. With amplicon sequencing the high number of different amplicons resulting from amplification of a usual sample are concatenated and sequenced. After quality control classification is done by different methods, the counts of identical taxa representing their relative abundance in the sample.


PCR can be used to determine sex from a human DNA sample. [ 16 ]   The  loci  of  Alu element  insertion is selected, amplified and evaluated in terms of size of the fragment. The sex assay utilizes AluSTXa for the  X chromosome , AluSTYa for the  Y chromosome , or both AluSTXa and AluSTYa, to reduce the possibility of error to a negligible quantity. The inserted chromosome yields a large fragment when the  homologous region  is amplified. The males are distinguished as having two DNA amplicons present, while females have only a single amplicon. The kit adapted for carrying out the method includes a pair of primers to amplify the locus and optionally polymerase chain reaction reagents. [ 17 ]


LCR can be used to diagnose  tuberculosis . [ 18 ]  The sequence containing protein  antigen  B is targeted by four  oligonucleotide   primers —two for the  sense strand , and two for the  antisense strand . The primers bind adjacent to one another, forming a segment of double stranded DNA that once separated, can serve as a target for future rounds of replication. In this instance, the product can be detected via the  microparticle enzyme immunoassay (MEIA) .


"What is an amplicon? See examples of the different applications" .  YouTube  video. 13 November 2013.


Amplified rDNA (Ribosomal DNA) Restriction Analysis  is the extension of the technique of RFLP ( restriction fragment length polymorphism ) to the gene encoding the  small (16s) ribosomal subunit  of  bacteria . The technique involves an enzymatic amplification using primers directed at the conserved regions at the ends of the 16s gene, followed by digestion using tetracutter  Restriction enzymes . The pattern obtained is said to be representative of the species analysed. Patterns obtained from several restriction enzymes can be used to phylogenetically characterize cultured isolates and 16s genes obtained through cloning from community  DNA [ 1 ]


Vaneechoutte  et al. , [ 2 ]  were among the first to use this method and applied it to characterize Mycobacterium species and Acinetobacter species  [ 3 ]  Numerous other studies have applied ARDRA for different bacterial taxa. Based on the simple formula for the frequency of random occurrence of a Restriction site, a 4-bp sequence can occur once every 256 bp. A restriction enzyme having a recognition site of more than 4 bp would be irrelevant with respect to a gene of approximately 1500 bp such as that coding for the 16s ribosomal subunit. A number of tetracutter Restriction enzymes are available in the market. For the sake of statistical significance, at least three restriction enzymes must be used for the analysis to overcome the probability of certain restriction enzymes to yield similar patterns for not so unrelated organisms.


The amplicon to be analysed must preferably correspond to a size of greater than 1000 bp, purely for the sake of encountering a greater possibility of the restriction site. The amplicons must be preferably purified before digestion. This can be done by numerous commercially available purification kits. Care should be taken to choose the restriction enzymes. Certain restriction enzymes recognize the same sites, and cannot contribute productively to the analysis. Overnight digestion (10–16 hours) of about 300-500 ng of amplicon DNA in a 20 μL system with 4-5 units of Restriction Enzyme along with the recommended buffer at the prescribed temperature is recommended. Following digestion, the reaction is stopped and the entire digest run in a 2-3% agarose gel at 90-100 V. The gel should be of a length that would allow proper resolution of minor fragments, as small as 100–200 bp.


Analysis of the patterns is done with methods used for  RAPD  patterns. Clusters of related bacteria can be represented in the form of a  cladogram  or  phylogram . After initial analysis a multivariate analysis program such as NT-SYS [ 4 ]  or PAST [ 5 ]  furnishing details about the presence or absence of bands, marked as 1(for presence) and 0(for absence). The data can be subsequently used for generating a  phylogram  or  cladogram . The data table can be used to plot a phylogenetic tree that would indicate the relationship of the organisms based on the restriction pattern obtained from their respective 16s genes. Commercial software is also available for the analysis of these patterns, most used are the packages GelCompar II and  BioNumerics .


Ancient DNA  ( aDNA ) is  DNA  isolated from ancient sources (typically  specimens , but also  environmental DNA ). [ 1 ] [ 2 ]  Due to degradation processes (including  cross-linking ,  deamination  and  fragmentation ) [ 3 ]  ancient DNA is more degraded in comparison with contemporary genetic material. [ 4 ]  Genetic material has been recovered from paleo/archaeological and historical skeletal material,  mummified  tissues, archival collections of non-frozen medical specimens, preserved plant remains, ice and from  permafrost  cores, marine and lake sediments and  excavation  dirt.


Even under the best preservation conditions, there is an upper boundary of 0.4–1.5 million years for a sample to contain sufficient DNA for sequencing technologies. [ 5 ]  The oldest DNA sequenced from physical specimens are from  mammoth  molars in Siberia over 1 million years old. [ 6 ]  In 2022, two-million year old genetic material was recovered from sediments in  Greenland , and is currently considered the oldest DNA discovered so far. [ 7 ] [ 8 ]


The first study of what would come to be called aDNA was conducted in 1984, when Russ Higuchi and colleagues at the  University of California, Berkeley  reported that traces of DNA from a museum specimen of the  Quagga  not only remained in the specimen over 150 years after the death of the individual, but could be extracted and sequenced. [ 9 ]  Over the next two years, through investigations into natural and artificially mummified specimens,  Svante Pääbo  confirmed that this phenomenon was not limited to relatively recent museum specimens but could apparently be replicated in a range of  mummified  human samples that dated as far back as several thousand years. [ 10 ] [ 11 ] [ 12 ]


The laborious processes that were required at that time to sequence such DNA (through  bacterial cloning ) were an effective brake on the study of ancient DNA (aDNA) and the field of  museomics . However, with the development of the  Polymerase Chain Reaction  (PCR) in the late 1980s, the field began to progress rapidly. [ 13 ] [ 14 ] [ 15 ]  Double primer PCR amplification of aDNA (jumping-PCR) can produce highly skewed and non-authentic sequence artifacts. Multiple primer,  nested PCR  strategy was used to overcome those shortcomings.


The post-PCR era heralded a wave of publications as numerous research groups claimed success in isolating aDNA. Soon a series of incredible findings had been published, claiming authentic DNA could be extracted from specimens that were millions of years old, into the realms of what Lindahl (1993b) has labelled  Antediluvian  DNA. [ 16 ]  The majority of such claims were based on the retrieval of DNA from organisms preserved in  amber . Insects such as stingless bees, [ 17 ] [ 18 ]  termites, [ 19 ]  and wood gnats, [ 20 ]  as well as plant [ 21 ]  and bacterial [ 22 ]  sequences were said to have been extracted from  Dominican  amber dating to the  Oligocene  epoch. Still older sources of Lebanese amber-encased  weevils , dating to within the  Cretaceous  epoch, reportedly also yielded authentic DNA. [ 23 ]  Claims of DNA retrieval were not limited to amber.


Reports of several sediment-preserved plant remains dating to the  Miocene  were published. [ 24 ] [ 25 ]  Then in 1994, Woodward  et al.  reported what at the time was called the most exciting results to date [ 26 ]  — mitochondrial cytochrome b sequences that had apparently been extracted from dinosaur bones dating to more than 80 million years ago. When in 1995 two further studies reported dinosaur DNA sequences extracted from a Cretaceous egg, [ 27 ] [ 28 ]  it seemed that the field would revolutionize knowledge of the Earth's evolutionary past. Even these extraordinary ages were topped by the claimed retrieval of 250-million-year-old halobacterial sequences from  halite . [ 29 ] [ 30 ]


The development of a better understanding of the kinetics of DNA preservation, the risks of sample contamination and other complicating factors led the field to view these results more skeptically. Numerous careful attempts failed to replicate many of the findings, and all of the decade's claims of multi-million year old aDNA would come to be dismissed as inauthentic. [ 31 ]


Single primer extension amplification was introduced in 2007 to address postmortem DNA modification damage. [ 32 ]  Since 2009 the field of aDNA studies has been revolutionized with the introduction of much cheaper research techniques. [ 33 ]  The use of high-throughput  Next Generation Sequencing  (NGS) techniques in the field of ancient DNA research has been essential for reconstructing the genomes of ancient or extinct organisms. A single-stranded DNA (ssDNA) library preparation method has sparked great interest among ancient DNA (aDNA) researchers. [ 34 ] [ 35 ]


In addition to these technical innovations, the start of the decade saw the field begin to develop better standards and criteria for evaluating DNA results, as well as a better understanding of the potential pitfalls. [ 31 ] [ 36 ]


Autumn of 2022, the Nobel Prize of Physiology or Medicine was awarded to Svante Pääbo "for his discoveries concerning the genomes of extinct hominins and human evolution". [ 37 ]  A few days later, on the 7th of December 2022, a study in  Nature  reported that two-million year old genetic material was found in Greenland, and is currently considered the oldest DNA discovered so far. [ 7 ] [ 8 ]


Due to degradation processes (including cross-linking, deamination and fragmentation), [ 3 ]  ancient DNA is of lower quality than modern genetic material. [ 4 ]  The damage characteristics and ability of aDNA to survive through time restricts possible analyses and places an upper limit on the age of successful samples. [ 4 ]  There is a theoretical correlation between time and DNA degradation, [ 38 ]  although differences in environmental conditions complicate matters. Samples subjected to different conditions are unlikely to predictably align to a uniform age-degradation relationship. [ 39 ]  The environmental effects may even matter after excavation, as DNA decay-rates may increase, [ 40 ]  particularly under fluctuating storage conditions. [ 41 ]  Even under the best preservation conditions, there is an upper boundary of 0.4 to 1.5 million years for a sample to contain sufficient DNA for contemporary sequencing technologies. [ 5 ]


Research into the decay of  mitochondrial  and  nuclear DNA  in  moa  bones has modelled mitochondrial DNA degradation to an average length of 1  base pair  after 6,830,000 years at −5 °C. [ 4 ]   The decay kinetics have been measured by accelerated aging experiments, further displaying the strong influence of storage temperature and humidity on DNA decay. [ 42 ]  Nuclear DNA degrades at least twice as fast as mtDNA. Early studies that reported recovery of much older DNA, for example from  Cretaceous   dinosaur  remains, may have stemmed from contamination of the sample.


A critical review of ancient DNA literature through the development of the field highlights that few studies have succeeded in amplifying DNA from remains older than several hundred thousand years. [ 43 ]  A greater appreciation for the risks of environmental contamination and studies on the  chemical stability  of DNA have raised concerns over previously reported results. The alleged dinosaur DNA was later revealed to be human  Y-chromosome . [ 44 ]  The DNA reported from encapsulated  halobacteria  has been criticized based on its similarity to modern bacteria, which hints at contamination, [ 36 ]  or they may be the product of long-term, low-level  metabolic  activity. [ 45 ]


aDNA may contain a large number of postmortem  mutations , increasing with time. Some regions of polynucleotide are more susceptible to this degradation, allowing erroneous sequence data to bypass statistical filters used to check the validity of data. [ 31 ]  Due to sequencing errors, great caution should be applied to interpretation of population size. [ 46 ]  Substitutions resulting from  deamination  of  cytosine  residues are vastly over-represented in the ancient DNA sequences. Miscoding of  C  to  T  and  G  to  A  accounts for the majority of errors. [ 47 ]


Another problem with ancient DNA samples is contamination by modern human DNA and by microbial DNA (most of which is also ancient). [ 48 ] [ 49 ]  New methods have emerged in recent years to prevent possible contamination of aDNA samples, including conducting extractions under extreme sterile conditions, using special adapters to identify endogenous molecules of the sample (distinguished from those introduced during analysis), and applying bioinformatics to resulting sequences based on known reads in order to approximate rates of contamination. [ 50 ] [ 51 ]


Development in the aDNA field in the 2000s increased the importance of authenticating recovered DNA to confirm that it is indeed ancient and not the result of recent contamination. As DNA degrades over time, the nucleotides that make up the DNA may change, especially at the ends of the DNA molecules. The deamination of cytosine to uracil at the ends of DNA molecules has become a way of authentication. During DNA sequencing, the DNA polymerases will incorporate an adenine (A) across from the uracil (U), leading to cytosine (C) to thymine (T) substitutions in the aDNA data. [ 52 ]  These substitutions increase in frequency as the sample gets older. Frequency measurement of the C-T level, ancient DNA damage, can be made using various software such as mapDamage2.0 or PMDtools  [ 53 ] [ 54 ]  and interactively on metaDMG. [ 55 ]  Due to hydrolytic depurination, DNA fragments into smaller pieces, leading to single-stranded breaks. Combined with the damage pattern, this short fragment length can also help differentiate between modern and ancient DNA. [ 56 ] [ 57 ]


Despite the problems associated with 'antediluvian' DNA, a wide and ever-increasing range of aDNA sequences have now been published from a range of animal and plant  taxa . Tissues examined include artificially or naturally mummified animal remains, [ 9 ] [ 58 ]  bone, [ 59 ] [ 60 ] [ 61 ] [ 62 ]  shells, [ 63 ]  paleofaeces, [ 64 ] [ 65 ]  alcohol preserved specimens, [ 66 ]  rodent middens, [ 67 ]  dried plant remains, [ 68 ] [ 69 ]  and recently, extractions of animal and plant DNA directly from  soil  samples. [ 70 ]


In June 2013, a group of researchers including  Eske Willerslev ,  Marcus Thomas Pius Gilbert  and Orlando Ludovic of the  Centre for Geogenetics ,  Natural History Museum of Denmark  at the  University of Copenhagen , announced that they had sequenced the DNA of a 560–780 thousand year old horse, using material extracted from a leg bone found buried in  permafrost  in Canada's  Yukon  territory. [ 71 ] [ 72 ] [ 73 ]  A German team also reported in 2013 the reconstructed  mitochondrial genome  of a bear,  Ursus deningeri , more than 300,000 years old, proving that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. [ 74 ]   The DNA sequence of even older nuclear DNA was reported in 2021 from the permafrost-preserved teeth of two Siberian  mammoths , both over a million years old. [ 6 ] [ 75 ]


Researchers in 2016 measured chloroplast DNA in marine sediment cores, and found diatom DNA dating back to 1.4 million years. [ 76 ]  This DNA had a half-life significantly longer than previous research, of up to 15,000 years. Kirkpatrick's team also found that DNA only decayed along a half-life rate until about 100 thousand years, at which point it followed a slower, power-law decay rate. [ 76 ]


Due to the considerable  anthropological ,  archaeological , and  public interest  directed toward human remains, they have received considerable attention from the DNA community.  There are also more profound contamination issues, since the specimens belong to the same species as the researchers collecting and evaluating the samples.


Due to the  morphological  preservation in mummies, many studies from the 1990s and 2000s used mummified tissue as a source of ancient human DNA. Examples include both naturally preserved specimens, such as the  Ötzi the Iceman  frozen in a glacier [ 78 ]  and bodies preserved through rapid  desiccation  at high altitude in the Andes, [ 12 ] [ 79 ]  as well as various chemically treated preserved tissue such as the mummies of ancient Egypt. [ 80 ]  However, mummified remains are a limited resource. The majority of human aDNA studies have focused on extracting DNA from two sources much more common in the  archaeological record :  bones  and  teeth . The bone that is most often used for DNA extraction is the  petrous  ear bone, since its dense structure provides good conditions for DNA preservation. [ 81 ]  Several other sources have also yielded DNA, including  paleofaeces , [ 82 ]  and  hair . [ 83 ] [ 84 ]  Contamination remains a major problem when working on ancient human material.


Ancient  pathogen  DNA has been successfully retrieved from samples dating to more than 5,000 years old in humans and as long as 17,000 years ago in other species. In addition to the usual sources of mummified tissue, bones and teeth, such studies have also examined a range of other tissue samples, including calcified  pleura , [ 85 ]  tissue embedded in  paraffin , [ 86 ] [ 87 ]  and  formalin -fixed tissue. [ 88 ]  Efficient computational tools have been developed for pathogen and microorganism aDNA analyses in a small (QIIME [ 89 ] ) and large scale (FALCON  [ 90 ] ).


Taking preventative measures in their procedure against such contamination though, a 2012 study analyzed bone samples of a  Neanderthal  group in the El Sidrón cave, finding new insights on potential kinship and genetic diversity from the aDNA. [ 91 ]  In November 2015, scientists reported finding a 110,000-year-old tooth containing DNA from the  Denisovan hominin , an  extinct   species  of  human  in the genus  Homo . [ 92 ] [ 93 ]


The research has added new complexity to the peopling of Eurasia. A study from 2018  [ 94 ]  showed that a  Bronze Age  mass migration had greatly impacted the genetic makeup of the British Isles, bringing with it the  Bell Beaker  culture from mainland Europe.


It has also revealed new information about links between the ancestors of Central Asians and the indigenous peoples of the Americas. In Africa, older DNA degrades quickly due to the warmer tropical climate, although, in September 2017, ancient DNA samples, as old as 8,100 years old, have been reported. [ 95 ]


Moreover, ancient DNA has helped researchers to estimate modern human divergence. [ 96 ]  By sequencing African genomes from three Stone Age hunter gatherers (2000 years old) and four Iron Age farmers (300 to 500 years old), Schlebusch and colleagues were able to push back the date of the earliest divergence between human populations to 350,000 to 260,000 years ago.


As of 2021, the oldest completely reconstructed human genomes are  ~45,000 years old . [ 97 ] [ 77 ]  Such genetic data provides insights into the migration and genetic history – e.g.  of Europe  – including about  interbreeding between archaic and modern humans  like a common admixture between initial European modern humans and Neanderthals. [ 98 ] [ 77 ] [ 99 ]


Aptamers  are  oligomers  of artificial  ssDNA ,  RNA ,  XNA , or  peptide  that  bind a specific target molecule , or family of target molecules. They exhibit a range of affinities ( K D  in the pM to μM range), [ 1 ] [ 2 ]  with variable levels of  off-target  binding [ 3 ]  and are sometimes classified as  chemical antibodies . Aptamers and antibodies can be used in many of the same applications, but the  nucleic acid -based structure of aptamers, which are mostly  oligonucleotides , is very different from the  amino acid -based structure of antibodies, which are  proteins . This difference can make aptamers a better choice than antibodies for some purposes (see  antibody replacement ).


Aptamers are used in biological lab research and  medical tests . If multiple aptamers are combined into a single  assay , they can  measure large numbers of different proteins in a sample . They can be used to  identify molecular markers of disease , or can function as  drugs ,  drug delivery systems  and  controlled drug release systems . They also find use in other  molecular engineering  tasks.


Most aptamers originate from  SELEX , a family of  test-tube experiments  for finding useful aptamers in a massive pool of different DNA sequences. This process is much like  natural selection ,  directed evolution  or artificial selection. In SELEX, the researcher repeatedly selects for the best aptamers from a starting DNA library made of about a  quadrillion  different  randomly generated pieces of DNA or RNA . After SELEX, the researcher might  mutate  or  change the chemistry  of the aptamers and do another selection, or might use  rational design  processes to engineer improvements.  Non-SELEX methods  for discovering aptamers also exist.


Researchers optimize aptamers to achieve a variety of beneficial features. The most important feature is  specific and sensitive  binding to the chosen target. When aptamers are exposed to bodily fluids, as in  serum  tests or aptamer therapeutics, it is often important for them to resist  digestion  by  DNA- and RNA-destroying proteins . Therapeutic aptamers often must be modified to  clear slowly from the body . Aptamers that  change their shape dramatically  when they bind their target are useful as  molecular switches  to turn a sensor on and off. Some aptamers are engineered to fit into a  biosensor  or in a  test of a biological sample . It can be useful in some cases for the aptamer to accomplish a pre-defined  level  or  speed  of binding. As the yield of the  synthesis  used to produce known aptamers shrinks quickly for longer sequences, [ 4 ]  researchers often truncate aptamers to the minimal binding sequence to reduce the production cost.


The word "aptamer" is a  neologism  coined by Andrew Ellington and Jack Szostak in their first publication on the topic. They did not provide a precise definition, stating "We have termed these individual RNA sequences 'aptamers', from the  Latin  ' aptus ', to fit." [ 5 ]


The word itself, however, derives from the  Greek  word  ἅπτω , to connect or fit (as used by Homer (c. 8th century BC) [ 6 ]   [ 7 ] ) and  μέρος,  a component of something larger. [ 8 ]


A typical aptamer is a synthetically generated  ligand  exploiting the  combinatorial diversity  of DNA, RNA, XNA, or peptide to achieve strong, specific binding for a particular target molecule or family of target molecules. Aptamers are occasionally classified as  "chemical antibodies" or "antibody mimics" . [ 9 ]  However, most aptamers are small, with a molecular weight of 6-30 kDa, in contrast to the 150 kDa size of antibodies, and contain one binding site rather than the two matching antigen binding regions of a typical antibody.


Since its first application in 1967, [ 10 ]  directed evolution methodologies have been used to develop  biomolecules  with new properties and functions. Early examples include the modification of the  bacteriophage Qbeta  replication system and the generation of  ribozymes  with modified  cleavage  activity. [ 11 ]


In 1990, two teams independently developed and published SELEX ( Systematic Evolution of Ligands by EXponential enrichment ) methods and generated RNA aptamers: the lab of Larry Gold, using the term SELEX for their process of selecting RNA  ligands  against T4  DNA polymerase [ 12 ]  and the lab of  Jack Szostak , selecting RNA ligands against various  organic dyes . [ 5 ] [ 13 ]  Two years later, the Szostak lab and  Gilead Sciences , acting independently of one another, used  in vitro  selection schemes to generate DNA aptamers for organic dyes [ 14 ]  and human  thrombin , [ 15 ]  respectively. In 2001, SELEX was automated by J. Colin Cox in the Ellington lab, reducing the duration of a weeks-long selection experiment to just three days. [ 16 ] [ 17 ] [ 18 ]


In 2002, two groups led by  Ronald Breaker  and  Evgeny Nudler  published the first definitive evidence for a  riboswitch , a nucleic acid-based  genetic regulatory element , the existence of which had previously been suspected. Riboswitches possess similar molecular recognition properties to aptamers. This discovery added support to the  RNA World hypothesis , a postulated stage in time in the  origin of life on Earth . [ 19 ]


Most aptamers are based on a specific oligomer sequence of 20-100  bases  and 3-20  kDa . Some have chemical modifications for functional enhancements or compatibility with larger engineered molecular systems. DNA, RNA,  XNA , and  peptide  aptamer chemistries can each offer distinct profiles in terms of shelf stability, durability in  serum  or  in vivo , specificity and sensitivity, cost, ease of generation, amplification, and characterization, and familiarity to users. Typically, DNA- and RNA-based aptamers exhibit low  immunogenicity , are amplifiable via  Polymerase Chain Reaction (PCR) , and have complex  secondary structure  and  tertiary structure . [ 20 ] [ 21 ] [ 22 ] [ 23 ]  DNA- and XNA-based aptamers exhibit superior shelf stability. XNA-based aptamers can introduce additional chemical diversity to increase  binding affinity  or greater durability in serum or  in vivo .


As 22 genetically-encoded and over 500 naturally-occurring  amino acids  exist,  peptide aptamers , as well as antibodies, have much greater potential combinatorial diversity per unit length relative to the 4 nucleic acids in DNA or RNA. [ 24 ]  Chemical modifications of nucleic acid bases or backbones increase the chemical diversity of standard nucleic acid bases. [ 25 ]


Split aptamers are composed of two or more DNA strands that are pieces of a larger parent aptamer that has been broken in two by a molecular  nick . [ 26 ]  The ability of each component strand to bind targets will depend on the location of the nick, as well as the secondary structures of the daughter strands. [ 27 ]  The presence of a target molecule supports the joining of DNA fragments. This can be used as the basis for biosensors. [ 28 ]  Once assembled, the two separate DNA strands can be  ligated  into a single strand.


Unmodified aptamers are cleared rapidly from the  bloodstream , with a  half-life  of seconds to hours. This is mainly due to  nuclease  degradation, which physically destroys the aptamers, as well as clearance by the  kidneys , a result of the aptamer's low  molecular weight  and size. Several modifications, such as 2'-fluorine-substituted  pyrimidines  and  polyethylene glycol  (PEG) linkage, permit a serum half-life of days to weeks.  PEGylation  can add sufficient mass and size to prevent clearance by the kidneys  in vivo . Unmodified aptamers can treat  coagulation  disorders. The problem of clearance and nuclease digestion is diminished when they are applied to the  eye , where there is a lower concentration of nuclease and the rate of clearance is lower. [ 29 ]  Rapid clearance from serum can also be useful in some applications, such as  in vivo   diagnostic imaging . [ 30 ]


In a study on aptamers [ 31 ]  designed to bind with proteins associated with Ebola infection, a comparison was made among three aptamers isolated for their ability to bind the target protein EBOV sGP. Although these aptamers vary in both sequence and structure, they exhibit remarkably similar relative affinities for sGP from EBOV and SUDV, as well as EBOV GP1.2. Notably, these aptamers demonstrated a high degree of specificity for the GP gene products. One aptamer, in particular, proved effective as a recognition element in an electrochemical sensor, enabling the detection of sGP and GP1.2 in solution, as well as GP1.2 within a membrane context.The results of this research point to the intriguing possibility that certain regions on protein surfaces may possess aptatropic qualities. Identifying the key features of such sites, in conjunction with improved 3-D structural predictions for aptamers, holds the potential to enhance the accuracy of predicting aptamer interaction sites on proteins. This, in turn, may help identify aptamers with a heightened likelihood of binding proteins with high affinity, as well as shed light on protein mutations that could significantly impact aptamer binding.This comprehensive understanding of the structure-based interactions between aptamers and proteins is vital for refining the computational predictability of aptamer-protein binding. Moreover, it has the potential to eventually eliminate the need for the experimental SELEX protocol.


Aptamer targets can include small molecules and  heavy metal  ions, larger ligands such as proteins, and even whole cells. [ 32 ] [ 33 ]  These targets include  lysozyme , [ 34 ]   thrombin , [ 35 ] [ 36 ]  human immunodeficiency virus  trans-acting responsive element  ( HIV  TAR), [ 37 ]   hemin , [ 38 ]   interferon γ , [ 39 ]   vascular endothelial growth factor  (VEGF), [ 40 ] [ 41 ]   prostate specific antigen  (PSA), [ 42 ] [ 43 ]   dopamine , [ 44 ]  and the non-classical  oncogene ,  heat shock factor 1  (HSF1). [ 45 ]


Aptamers have been generated against cancer cells, [ 46 ]   prions , [ 47 ]  bacteria, [ 48 ]  and viruses. Viral targets of aptamers include  influenza A  and  B  viruses, [ 49 ]   Respiratory syncytial virus  (RSV), [ 49 ]   SARS coronavirus  (SARS-CoV) [ 49 ]  and  SARS-CoV-2 . [ 50 ]


Aptamers may be particularly useful for  environmental science  proteomics. [ 51 ]  Antibodies, like other proteins, are more difficult to sequence than nucleic acids. They are also costly to maintain and produce, and are at constant risk of contamination, as they are produced via  cell culture  or are harvested from animal serum. For this reason, researchers interested in little-studied proteins and species may find that companies will not produce, maintain, or adequately validate the quality of antibodies against their target of interest. [ 52 ]  By contrast, aptamers are simple to sequence and cost nothing to maintain, as their exact structure can be stored digitally and synthesized on demand. This may make them more economically feasible as research tools for underfunded biological research subjects. Aptamers exist for plant compounds, such as  theophylline  (found in  tea ) [ 53 ]  and  abscisic acid  (a plant immune hormone). [ 54 ]  An aptamer against  a-amanitin  (the toxin that causes lethal  Amanita  poisoning) has been developed, an example of an aptamer against a  mushroom  target. [ 55 ]


Aptamer applications can be roughly grouped into sensing, therapeutic, reagent production, and engineering categories. Sensing applications are important in environmental, biomedical,  epidemiological ,  biosecurity , and basic research applications, where aptamers act as probes in assays, imaging methods, diagnostic assays, and biosensors. [ 32 ] [ 56 ] [ 57 ] [ 58 ] [ 59 ] [ 60 ]  In therapeutic applications and  precision medicine , aptamers can function as drugs, [ 61 ]  as targeted  drug delivery  vehicles, [ 62 ]  as controlled release mechanisms, and as reagents for  drug discovery  via  high-throughput screening  for small molecules [ 63 ]  and proteins. [ 64 ] [ 65 ]  Aptamers have application for protein production monitoring, quality control, and purification. [ 66 ] [ 67 ] [ 68 ]  They can function in molecular engineering applications as a way to modify proteins, such as enhancing DNA polymerase to make PCR more reliable. [ 69 ] [ 70 ] [ 71 ] [ 72 ]


Because the affinity of the aptamer also affects its dynamic range and limit of detection, aptamers with a lower affinity may be desirable when assaying high concentrations of a target molecule. [ 73 ]  Affinity chromatography also depends on the ability of the affinity reagent, such as an aptamer, to bind and release its target, and lower affinities may aid in the release of the target molecule. [ 74 ]  Hence, specific applications determine the useful range for aptamer affinity.


Aptamers can replace antibodies in many  biotechnology  applications. [ 75 ] [ 52 ]  In laboratory research and clinical diagnostics, they can be used in aptamer-based versions of  immunoassays  including  enzyme-linked immunosorbent assay (ELISA) , [ 76 ]   western blot , [ 77 ]   immunohistochemistry (IHC) , [ 78 ]  and  flow cytometry . [ 79 ]  As therapeutics, they can function as  agonists  or  antagonists  of their ligand. [ 80 ]  While antibodies are a familiar technology with a well-developed market, aptamers are a relatively new technology to most researchers, and aptamers have been generated against only a fraction of important research targets. [ 81 ]  Unlike antibodies, unmodified aptamers are more susceptible to nuclease digestion in serum and renal clearance  in vivo . Aptamers are much smaller in size and mass than antibodies, which could be a relevant factor in choosing which is best suited for a given application. When aptamers are available for a particular application, their advantages over antibodies include potentially lower immunogenicity, greater  replicability  and lower cost, a greater level of control due to the  in vitro  selection conditions, and capacity to be efficiently engineered for durability, specificity, and sensitivity. [ 82 ]


In addition, aptamers contribute to  reduction of research animal use . [ 83 ]  While antibodies often rely on animals for initial discovery, as well as for production in the case of  polyclonal antibodies , both the selection and production of aptamers is typically animal-free. However,  phage display  methods allow for selection of antibodies  in vitro , followed by production from a  monoclonal  cell line, avoiding the use of animals entirely. [ 84 ]


The ability of aptamers to reversibly bind molecules such as proteins has generated increasing interest in using them to facilitate  controlled release  of therapeutic biomolecules, such as  growth factors . This can be accomplished by tuning the binding strength to passively release the growth factors, [ 85 ]  along with active release via mechanisms such as  hybridization  of the aptamer with complementary  oligonucleotides [ 86 ]  or unfolding of the aptamer due to cellular traction forces. [ 87 ]


Aptamer, known for their ability to bind specific molecules reversibly, have been used in  3D bioprinting  tissues to precisely deliver growth factors to promote  vascularization . [ 88 ] [ 89 ]  This controlled delivery allows  growth factors  to be released at the right place and time, encouraging the formation of localized and complex vascular networks. Additionally, the properties of these networks can be fine-tuned by adjusting how growth factors are released over time, making this approach a powerful tool for creating vascularized engineered tissues.


AptaBiD (Aptamer-Facilitated Biomarker Discovery) is an aptamer-based method for  biomarker  discovery. [ 90 ]


While most aptamers are based on DNA, RNA, or XNA, peptide aptamers [ 91 ]  are artificial proteins selected or engineered to bind specific target molecules.


Peptide aptamers consist of one or more peptide loops of variable sequence displayed by a protein scaffold.  Derivatives  known as tadpoles, in which peptide aptamer "heads" are covalently linked to unique sequence double-stranded DNA "tails", allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative  real-time polymerase chain reaction ) of their DNA tails. [ 92 ]  The peptides that form the aptamer variable regions are synthesized as part of the same polypeptide chain as the scaffold and are constrained at their  N  and  C termini  by linkage to it. This double structural constraint decreases the diversity of the 3D structures that the variable regions can adopt, [ 93 ]  and this reduction in structural diversity lowers the entropic cost of  molecular binding  when interaction with the target causes the variable regions to adopt a uniform structure.


The most common peptide aptamer selection system is the  yeast   two-hybrid system . Peptide aptamers can also be selected from combinatorial  peptide libraries  constructed by  phage display  and other surface display technologies such as  mRNA display ,  ribosome display ,  bacterial display  and  yeast display . These experimental procedures are also known as  biopanning . All the peptides panned from combinatorial peptide libraries have been stored in the  MimoDB  database. [ 94 ] [ 95 ]


Libraries of peptide aptamers have been used as "mutagens", in studies in which an investigator introduces a library that expresses different peptide aptamers into a cell population, selects for a desired  phenotype , and identifies those aptamers that cause the phenotype. The investigator then uses those aptamers as baits, for example in yeast two-hybrid screens to identify the cellular proteins targeted by those aptamers. Such experiments identify particular proteins bound by the aptamers, and protein interactions that the aptamers disrupt, to cause the phenotype. [ 96 ] [ 97 ]  In addition, peptide aptamers derivatized with appropriate functional  moieties  can cause specific  post-translational modification  of their target proteins, or change the subcellular localization of the targets. [ 98 ]


Commercial products and companies based on aptamers include the drug  Macugen (pegaptanib) [ 99 ]  and the  clinical diagnostic  company SomaLogic. [ 100 ]  The International Society on Aptamers (INSOAP), a professional society for the aptamer research community, publishes a  journal  devoted to the topic,  Aptamers . Apta-index [ 101 ]  is a current database cataloging and simplifying the ordering process for over 700 aptamers.


Artificial gene synthesis , or simply  gene synthesis , refers to a group of methods that are used in  synthetic biology  to construct and assemble  genes  from  nucleotides   de novo . Unlike  DNA  synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is  solid-phase DNA synthesis , sometimes known as  DNA printing . [ 1 ]  This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.


Synthesis of the first complete gene, a yeast  tRNA , was demonstrated by  Har Gobind Khorana  and coworkers in 1972. [ 2 ]  Synthesis of the first  peptide - and  protein -coding genes was performed in the laboratories of  Herbert Boyer  and  Alexander Markham , respectively. [ 3 ] [ 4 ]  More recently, artificial gene synthesis methods have been developed that will allow the assembly of entire chromosomes and genomes. The first synthetic  yeast  chromosome was synthesised in 2014, and entire functional  bacterial  chromosomes have also been synthesised. [ 5 ]  In addition, artificial gene synthesis could in the future make use of novel  nucleobase  pairs (unnatural base pairs). [ 6 ] [ 7 ] [ 8 ]


Oligonucleotides  are chemically synthesized using building blocks called nucleoside  phosphoramidites . These can be normal or modified nucleosides which have protecting groups to prevent their amines, hydroxyl groups and phosphate groups from interacting incorrectly. One phosphoramidite is added at a time, the 5' hydroxyl group is deprotected and a new base is added and so on. The chain grows in the 3' to 5' direction, which is backwards relative to biosynthesis. At the end, all the protecting groups are removed. Nevertheless, being a chemical process, several incorrect interactions occur leading to some defective products. The longer the oligonucleotide sequence that is being synthesized, the more defects there are, thus this process is only practical for producing short sequences of  nucleotides . The current practical limit is about 200 bp ( base pairs ) for an oligonucleotide with sufficient quality to be used directly for a biological application.  HPLC  can be used to isolate products with the proper sequence. Meanwhile, a large number of oligos can be synthesized in parallel on  gene chips . For optimal performance in subsequent gene synthesis procedures they should be prepared individually and in larger scales.


Usually, a set of individually designed oligonucleotides is made on automated solid-phase synthesizers, purified and then connected by specific  annealing  and standard  ligation  or  polymerase  reactions. To improve specificity of oligonucleotide annealing, the synthesis step relies on a set of thermostable DNA  ligase  and  polymerase   enzymes . To date, several methods for gene synthesis have been described, such as the ligation of phosphorylated overlapping oligonucleotides, [ 2 ] [ 3 ]  the Fok I method [ 4 ]  and a modified form of  ligase chain reaction  for gene synthesis. Additionally, several  PCR  assembly approaches have been described. [ 9 ]  They usually employ oligonucleotides of 40-50 nucleotides length that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension (OE) PCR, [ 9 ]  thermodynamically balanced inside-out (TBIO) PCR [ 10 ]  or combined approaches. [ 11 ]  The most commonly synthesized genes range in size from 600 to 1,200 bp although much longer genes have been made by connecting previously assembled fragments of under 1,000 bp. In this size range it is necessary to test several candidate clones confirming the sequence of the cloned synthetic gene by automated sequencing methods.


Moreover, because the assembly of the full-length gene product relies on the efficient and specific alignment of long single stranded oligonucleotides, critical parameters for synthesis success include extended sequence regions comprising secondary structures caused by inverted repeats, extraordinary high or low GC-content, or repetitive structures. Usually these segments of a particular gene can only be synthesized by splitting the procedure into several consecutive steps and a final assembly of shorter sub-sequences, which in turn leads to a significant increase in time and labor needed for its production.
The result of a gene synthesis experiment depends strongly on the quality of the oligonucleotides used. For these annealing based gene synthesis protocols, the quality of the product is directly and exponentially dependent on the correctness of the employed oligonucleotides. Alternatively, after performing gene synthesis with oligos of lower quality, more effort must be made in downstream quality assurance during clone analysis, which is usually done by time-consuming standard cloning and sequencing procedures.
Another problem associated with all current gene synthesis methods is the high frequency of sequence errors because of the usage of chemically synthesized oligonucleotides. The error frequency increases with longer oligonucleotides, and as a consequence the percentage of correct product decreases dramatically as more oligonucleotides are used.
The mutation problem could be solved by shorter oligonucleotides used to assemble the gene. However, all annealing based assembly methods require the primers to be mixed together in one tube. In this case, shorter overlaps do not always allow precise and specific annealing of complementary primers, resulting in the inhibition of full length product formation.
Manual design of oligonucleotides is a laborious procedure and does not guarantee the successful synthesis of the desired gene. For optimal performance of almost all annealing based methods, the melting temperatures of the overlapping regions are supposed to be similar for all oligonucleotides. The necessary primer optimisation should be performed using specialized oligonucleotide design programs. Several solutions for automated primer design for gene synthesis have been presented so far. [ 12 ] [ 13 ] [ 14 ]


To overcome problems associated with oligonucleotide quality several elaborate strategies have been developed, employing either separately prepared fishing oligonucleotides, [ 15 ]  mismatch binding enzymes of the  mutS family [ 16 ]  or specific endonucleases from bacteria or phages. [ 17 ]  Nevertheless, all these strategies increase time and costs for gene synthesis based on the annealing of chemically synthesized oligonucleotides.


Massively parallel sequencing has also been used as a tool to screen complex oligonucleotide libraries and enable the retrieval of accurate molecules. In one approach, oligonucleotides are sequenced on the 454 pyrosequencing platform and a robotic system images and picks individual beads corresponding to accurate sequence. [ 18 ]  In another approach, a complex oligonucleotide library is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by dial-out PCR. [ 19 ]


Increasingly, genes are ordered in sets including functionally related genes or multiple sequence variants on a single gene. Virtually all of the therapeutic proteins in development, such as monoclonal antibodies, are optimised by testing many gene variants for improved function or expression.


While traditional nucleic acid synthesis only uses 4 base pairs - adenine, thymine, guanine and cytosine, oligonucleotide synthesis in the future could incorporate the use of unnatural base pairs, which are artificially designed and synthesized nucleobases that do not occur in nature.