Polymerase chain reaction

Source: Wikipedia, the free encyclopedia.
(Redirected from
PCR testing
)

A strip of eight PCR tubes, each containing a 100 μL reaction mixture
Placing a strip of eight PCR tubes into a thermal cycler

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.[1]

PCR is fundamental to many of the procedures used in

biomedical research and forensic science.[2][3]

The majority of PCR methods rely on

DNA melting and enzyme-driven DNA replication. PCR employs two main reagents—primers (which are short single strand DNA fragments known as oligonucleotides that are a complementary sequence to the target DNA region) and a DNA polymerase. In the first step of PCR, the two strands of the DNA double helix are physically separated at a high temperature in a process called nucleic acid denaturation. In the second step, the temperature is lowered and the primers bind to the complementary sequences of DNA. The two DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially
amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.[4]

Applications of the technique include

infectious diseases
.

Principles

An older, three-temperature thermal cycler for PCR

PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10

kilo base pairs (kbp) in length, although some techniques allow for amplification of fragments up to 40 kbp.[6] The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the reaction progresses.[7]

A basic PCR set-up requires several components and reagents,[8] including:

The reaction is commonly carried out in a volume of 10–200 

thermal conductivity to allow for rapid thermal equilibrium. Most thermal cyclers have heated lids to prevent condensation
at the top of the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

Typically, PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps (see figure below). The cycling is often preceded by a single temperature step at a very high temperature (>90 °C (194 °F)), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the

melting temperature (Tm) of the primers.[12]
The individual steps common to most PCR methods are as follows:

It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of the primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very closely matches the template sequence. During this step, the polymerase binds to the primer-template hybrid and begins DNA formation.
The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles. Thus, a reaction set for 30 cycles results in 230, or 1,073,741,824, copies of the original double-stranded DNA target region.
  • Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
  • Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-term storage of the PCR products.
Schematic drawing of a complete PCR cycle
Schematic drawing of a complete PCR cycle
Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.

To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or

DNA ladder
, a molecular weight marker which contains DNA fragments of known sizes, which runs on the gel alongside the PCR products.

Tucker PCR
Tucker PCR

Stages

Exponential amplification

As with other chemical reactions, the reaction rate and efficiency of PCR are affected by limiting factors. Thus, the entire PCR process can further be divided into three stages based on reaction progress:

  • Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 (one billion) copies. In a sense, then, the replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions.[16] The reaction is very sensitive: only minute quantities of DNA must be present.
  • Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited.
  • Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

Optimization

In practice, PCR can fail for various reasons, such as sensitivity or contamination.[17][18] Contamination with extraneous DNA can lead to spurious products and is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[8] For instance, if DNA from a crime scene is analyzed, a single DNA molecule from lab personnel could be amplified and misguide the investigation. Hence the PCR-setup areas is separated from the analysis or purification of other PCR products, disposable plasticware used, and the work surface between reaction setups needs to be thoroughly cleaned.

Specificity can be adjusted by experimental conditions so that no spurious products are generated. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of unspecific products. The usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. For instance, Q5 polymerase is said to be ~280 times less error-prone than Taq polymerase.[19][20] Both the running parameters (e.g. temperature and duration of cycles), or the addition of reagents, such as formamide, may increase the specificity and yield of PCR.[21] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.[22]

Applications

Selective DNA isolation

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many ways, such as generating

DNA cloning
, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include

genetic fingerprinting
; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Electrophoresis of PCR-amplified DNA fragments:
  1. Father
  2. Child
  3. Mother

The child has inherited some, but not all, of the fingerprints of each of its parents, giving it a new, unique fingerprint.

Some PCR fingerprint methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms when certain

16S rRNA and recA genes of microorganisms).[24]

Amplification and quantification of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for

Richard III.[25]

Quantitative PCR or Real Time PCR (qPCR,[26] not to be confused with RT-PCR) methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression
. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA (cDNA) takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique, called RT-qPCR, allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR,[27] increasing chances for detection of genes associated with genetic diseases such as cancer.[5] Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the PCR[28] and RT-qPCR[29] facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications.[30][31][32][33]

Medical and diagnostic applications

Prospective parents can be tested for being

prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to preimplantation genetic diagnosis
, where individual cells of a developing embryo are tested for mutations.

  • PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of 2008, there is even a proposal to replace the traditional antibody-based tests for blood type with PCR-based tests.[34]
  • Many forms of cancer involve alterations to
    malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods.[35] PCR is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors. Quantitative PCR for example, can be used to quantify and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and combinations.[27]

Infectious disease applications

PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.

animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.[36][37]

Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:

  • The human immunodeficiency virus (or
    tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells.[38] Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified
    .
  • Some disease organisms, such as that for tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated.
  • The spread of a
    earlier epidemics
    can also be determined by PCR analysis.
  • Viral DNA can be detected by PCR. The primers used must be specific to the targeted sequences in the DNA of a virus, and PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease.[36] Such early detection may give physicians a significant lead time in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below). A variant of PCR (RT-PCR) is used for detecting viral RNA rather than DNA: in this test the enzyme reverse transcriptase is used to generate a DNA sequence which matches the viral RNA; this DNA is then amplified as per the usual PCR method. RT-PCR is widely used to detect the SARS-CoV-2 viral genome.[39]
  • Diseases such as pertussis (or
    dimers and reacts with different cell types such as T lymphocytes which play a role in cell immunity.[40] PCR is an important testing tool that can detect sequences within the gene for the pertussis toxin. Because PCR has a high sensitivity for the toxin and a rapid turnaround time, it is very efficient for diagnosing pertussis when compared to culture.[41]

Forensic applications

The development of PCR-based

forensics
:

  • DNA database of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming or exonerating
    the people originally convicted.
  • Forensic DNA typing has been an effective way of identifying or exonerating criminal suspects due to analysis of evidence discovered at a crime scene. The human genome has many repetitive regions that can be found within gene sequences or in non-coding regions of the genome. Specifically, up to 40% of human DNA is repetitive.[5] There are two distinct categories for these repetitive, non-coding regions in the genome. The first category is called variable number tandem repeats (VNTR), which are 10–100 base pairs long and the second category is called short tandem repeats (STR) and these consist of repeated 2–10 base pair sections. PCR is used to amplify several well-known VNTRs and STRs using primers that flank each of the repetitive regions. The sizes of the fragments obtained from any individual for each of the STRs will indicate which alleles are present. By analyzing several STRs for an individual, a set of alleles for each person will be found that statistically is likely to be unique.[5] Researchers have identified the complete sequence of the human genome. This sequence can be easily accessed through the NCBI website and is used in many real-life applications. For example, the FBI has compiled a set of DNA marker sites used for identification, and these are called the Combined DNA Index System (CODIS) DNA database.[5] Using this database enables statistical analysis to be used to determine the probability that a DNA sample will match. PCR is a very powerful and significant analytical tool to use for forensic DNA typing because researchers only need a very small amount of the target DNA to be used for analysis. For example, a single human hair with attached hair follicle has enough DNA to conduct the analysis. Similarly, a few sperm, skin samples from under the fingernails, or a small amount of blood can provide enough DNA for conclusive analysis.[5]
  • Less discriminating forms of
    DNA fingerprinting can help in DNA paternity testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed
    (or ruled out).
  • The PCR AMGX/AMGY design has been shown to not only[clarification needed] facilitate in amplifying DNA sequences from a very minuscule amount of genome. However it can also be used for real-time sex determination from forensic bone samples. This provides a powerful and effective way to determine gender in forensic cases and ancient specimens.[42]

Research applications

PCR has been applied to many areas of research in molecular genetics:

  • PCR allows rapid production of short pieces of DNA, even when not more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generating hybridization probes for Southern or northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material.
  • The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest.
  • PCR has numerous applications to the more traditional process of
    DNA cloning
    . It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of 'vector primers', it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment.
  • Sequence-tagged sites is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone. The Human Genome Project found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.
  • An application of PCR is the
    phylogenic analysis of DNA from ancient sources, such as that found in the recovered bones of Neanderthals, from frozen tissues of mammoths, or from the brain of Egyptian mummies.[16]
    In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification.
  • A common application of PCR is the study of patterns of
    quantitative PCR
    to quantitate the actual levels of expression
  • The ability of PCR to simultaneously amplify several loci from individual sperm[43] has greatly enhanced the more traditional task of genetic mapping by studying chromosomal crossovers after meiosis. Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms. Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay) for the long and laborious processes of fertilization, embryogenesis, etc.
  • Site-directed mutagenesis: PCR can be used to create mutant genes with mutations chosen by scientists at will. These mutations can be chosen in order to understand how proteins accomplish their functions, and to change or improve protein function.

Advantages

PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. qRT-PCR shares the same advantages as the PCR, with an added advantage of quantification of the synthesized product. Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states.[27]

PCR is a very powerful and practical research tool. The sequencing of unknown etiologies of many diseases are being figured out by the PCR. The technique can help identify the sequence of previously unknown viruses related to those already known and thus give us a better understanding of the disease itself. If the procedure can be further simplified and sensitive non-radiometric detection systems can be developed, the PCR will assume a prominent place in the clinical laboratory for years to come.[16]

Limitations

One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification.[27] This means that, typically, PCR users must know the precise sequence(s) upstream of the target region on each of the two single-stranded templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids and subsequently generates the entire target region during DNA synthesis.

Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated.[44]

Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. To minimize the chance of contamination, investigators should reserve separate rooms for reagent preparation, the PCR, and analysis of product. Reagents should be dispensed into single-use aliquots. Pipettors with disposable plungers and extra-long pipette tips should be routinely used.[16] It is moreover recommended to ensure that the lab set-up follows a unidirectional workflow. No materials or reagents used in the PCR and analysis rooms should ever be taken into the PCR preparation room without thorough decontamination.[45]

Environmental samples that contain humic acids may inhibit PCR amplification and lead to inaccurate results.

Variations

History

Diagrammatic representation of an example primer pair. The use of primers in an in vitro assay to allow DNA synthesis was a major innovation that allowed the development of PCR.

The heat-resistant enzymes that are a key component in polymerase chain reaction were discovered in the 1960s as a product of a microbial life form that lived in the superheated waters of Yellowstone's Mushroom Spring.[81]

A 1971 paper in the Journal of Molecular Biology by Kjell Kleppe and co-workers in the laboratory of H. Gobind Khorana first described a method of using an enzymatic assay to replicate a short DNA template with primers in vitro.[82] However, this early manifestation of the basic PCR principle did not receive much attention at the time and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[83][page needed]

"Baby Blue", a 1986 prototype machine for doing PCR

When Mullis developed the PCR in 1983, he was working in

paternity testing in 1988.[86]

Mullis has credited his use of LSD as integral to his development of PCR: "Would I have invented PCR if I hadn't taken LSD? I seriously doubt it. I could sit on a DNA molecule and watch the polymers go by. I learnt that partly on psychedelic drugs."[87]

Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA,[1] were jointly awarded the Nobel Prize in Chemistry in 1993, seven years after Mullis and his colleagues at Cetus first put his proposal to practice.[88] Mullis's 1985 paper with R. K. Saiki and H. A. Erlich, "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia"—the polymerase chain reaction invention (PCR)—was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017.[89][2]

At the core of the PCR method is the use of a suitable

DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures.[2]
So the early procedures for DNA replication were very inefficient and time-consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.

The discovery in 1976 of Taq polymerase—a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments[14] such as hot springs—paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[15] thus obviating the need to add new DNA polymerase after each cycle.[3] This allowed an automated thermocycler-based process for DNA amplification.

Patent disputes

The PCR technique was patented by

Hoffmann-La Roche purchased the rights to the patents in 1992. The last of the commercial PCR patents expired in 2017.[91]

A related patent battle over the Taq polymerase enzyme is still ongoing[as of?] in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on 28 March 2005.[92]

See also

References

  1. ^ a b "The Nobel Prize in Chemistry 1993". NobelPrize.org.
  2. ^
    PMID 2999980
    .
  3. ^ .
  4. .
  5. ^ .
  6. .
  7. .
  8. ^ . Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
  9. ^ "Polymerase Chain Reaction (PCR)". National Center for Biotechnology Information, U.S. National Library of Medicine.
  10. ^ "PCR". Genetic Science Learning Center, University of Utah.
  11. PMID 15109812
    .
  12. .
  13. ^ .
  14. ^ .
  15. ^ .
  16. ^ .
  17. ^ Borman, Jon, Schuster, David, Li, Wu-bo, Jessee, Joel, Rashtchian, Ayoub (2000). "PCR from problematic templates" (PDF). Focus. 22 (1): 10. Archived from the original (PDF) on 7 March 2017.
  18. ^ Bogetto P, Waidne L, Anderson H (2000). "Helpful tips for PCR" (PDF). Focus. 22 (1): 12. Archived from the original (PDF) on 7 March 2017.
  19. ^ Biolabs NE. "Q5® High-Fidelity DNA Polymerase | NEB". www.neb.com. Retrieved 4 December 2021.
  20. PMID 31118299
    .
  21. .
  22. ^ "Electronic PCR". NCBI – National Center for Biotechnology Information. Retrieved 13 March 2012.
  23. .
  24. .
  25. ^ "Chemical Synthesis, Sequencing, and Amplification of DNA (class notes on MBB/BIO 343)". Arizona State University. Archived from the original on 9 October 1997. Retrieved 29 October 2007.
  26. PMID 19246619
    .
  27. ^ .
  28. .
  29. .
  30. .
  31. .
  32. .
  33. .
  34. .
  35. .
  36. ^ .
  37. .
  38. .
  39. ^ "Coronavirus: il viaggio dei test". Istituto Superiore di Sanità.
  40. PMID 21413270
    .
  41. .
  42. .
  43. .
  44. .
  45. ^ Stursberg S (2021). "Setting up a PCR lab from scratch". INTEGRA Biosciences.
  46. S2CID 212693790
    .
  47. .
  48. .
  49. .
  50. .
  51. .
  52. .
  53. .
  54. .
  55. .
  56. .
  57. .
  58. .
  59. .
  60. .
  61. .
  62. .
  63. .
  64. .
  65. .
  66. .
  67. .
  68. ^ Fabrice David (September–October 2002). "Utiliser les propriétés topologiques de l'ADN: une nouvelle arme contre les agents pathogènes" (PDF). Fusion. Archived from the original (PDF) on 28 November 2007.(in French)
  69. PMID 23254127
    .
  70. .
  71. .
  72. ^ Bing DH, Boles C, Rehman FN, Audeh M, Belmarsh M, Kelley B, et al. (1996). "Bridge amplification: a solid phase PCR system for the amplification and detection of allelic differences in single copy genes". Genetic Identity Conference Proceedings, Seventh International Symposium on Human Identification. Archived from the original on 7 May 2001.
  73. PMID 18267099
    .
  74. .
  75. .
  76. .
  77. .
  78. ^ "Full Text – LaNe RAGE: a new tool for genomic DNA flanking sequence determination". www.ejbiotechnology.info. Archived from the original on 16 May 2008. Retrieved 24 April 2008.
  79. S2CID 45702164
    .
  80. .
  81. ^ "Key ingredient in coronavirus tests comes from Yellowstone's lakes". Science. 31 March 2020. Archived from the original on 31 March 2020. Retrieved 13 May 2020.
  82. PMID 4927950
    .
  83. .
  84. .
  85. .
  86. .
  87. .
  88. ^ "The Nobel Prize in Chemistry 1993". NobelPrize.org.
  89. ^ "Citations for Chemical Breakthrough Awards 2017 Awardees". Division of the History of Chemistry. Retrieved 12 March 2018.
  90. ^ "Recently added".
  91. PMID 16817955
    .
  92. ^ "Advice on How to Survive the Taq Wars". GEN Genetic Engineering News – Biobusiness Channel. 26 (9). 1 May 2006. Archived from the original on 26 March 2020. Retrieved 24 April 2019.

External links