Polymerase chain reaction

 

Polymerase chain reaction (PCR) is a molecular biology technique[1] for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.

PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.

PCR in practice

PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coded sequence. PCR typically amplifies only short DNA fragments, usually up to 10 kilo base pairs (kb). Certain methods can copy fragments up to 25 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell - for example, a human cell contains about three billion base pairs (3 Gbp).

PCR, as currently practiced, requires several basic components [3]. These components are:

  • DNA template, which contains the region of the DNA fragment to be amplified
  • Two primers, which determine the beginning and end of the region to be amplified (see following section on primers)
  • Taq polymerase (or another durable polymerase), a DNA polymerase which copies the region to be amplified
  • Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
  • Buffer solution, which provides a suitable chemical environment for the DNA Polymerase
  • Divalent cation, magnesium or manganese ions
  • Monovalent cation, potassium ions

The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. The lid of the thermal cycler is heated to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation. Typical reaction volumes range from 15-100 μl.

Primers

The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand.

The choice of the length of the primers and their melting temperature (Tm) depends on a number of considerations. The melting temperature of a primer -- not to be confused with the melting temperature of the template DNA -- is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C.

The above mentioned considerations make primer design a very exacting process, upon which product yield depends:

  • GC-content should be between 40-60%.
  • Calculated Tm for both primers used in reaction should not differ >5°C, and Tm of the amplification product should not differ from primers by >10°C (In practice may not be true always).
  • Annealing temperature usually is 5°C below the calculated lower Tm. However, it should be chosen empirically for individual conditions.
  • Inner self-complementary hairpins of >4 and of dimers >8 should be avoided.
  • Primer 3' terminus design is critical to PCR success since the primer extends from the 3' end. The 3' end should not be complementary over greater than 3-4 bases to any region of the other primer (or even the same primer) used in the reaction and must provide correct base matching to the template.

The animation below shows how PCR works:

 

Procedure

The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps.

  1. The double-stranded DNA has to be heated to 94-96°C (or 98°C if extremely thermostable polymerases are used) in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: usually 1-2 minutes, but up to 5 minutes. .
  2. After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-65°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 30 seconds-2 minutes.
  3. Finally, the DNA polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA polymerase. Taq polymerase elongates optimally at a temperature of 72 degrees Celsius. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, this step takes 1 minute per thousand base pairs. A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied.

Example.

Gel electrophoresis image of a standard PCR. Two sets of specific primers were used to amplify one gene from three separate tissues. As the gel shows, Tissue #1 lacks that gene, whereas Tissue #2 and #3 possess that gene.

The reaction mixture consists of

  • 1.0 µl DNA template (100 ng/µl)
  • 2.5 µl of primer, 1.25 µl per primer (100 ng/µl)
  • 1.0 µl Pfu-Polymerase
  • 1.0 µl nucleotides
  • 5.0 µl buffer solution
  • 89.5 µl water

A 200 µl reaction tube containing the 100 µl mixture is inserted into the thermocycler and cycled at 94oC - 55oC - 72oC 25 times..

The PCR product can be identified by its size using agarose gel electrophoresis. Agarose gel electrophoresis is a procedure that consists of injecting DNA into agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size, also within the gel (Fig. 3).

PCR optimization

Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in the lab environment (bacteria, viruses, lab staff's skin etc.) should be taken. Thus DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. For the preparation of reaction mixture, a laminar flow cabinet with UV lamp is recommended. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should be prepared separately and used solely for this purpose. Aliquots should be stored separately from other DNA samples. A control reaction (inner control), omitting template DNA, should always be performed, to confirm the absence of contamination or primer multimer formation.

Hairpins

Secondary structures in the DNA, caused by base-pairing between nucleotides on the same strand of the molecule, can cause folding or even knotting of the DNA template or the primers, leading to decreased yield or total failure of the reaction. Hairpins, direct folding of the DNA caused by a run of complementary bases or an inversion, are the most common problems of this sort.

Typically, this calls for choosing different primers; secondary structures in the template DNA are not as serious as those in the primers, as the DNA polymerase will "flatten out" most secondary structures unless they are particularly robust.

Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to check the base it has inserted and remove it if it is incorrect, a process common in higher organisms. This in turn results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early, can alter large proportions of the final product.

Other polymerases are available for accuracy in vital uses such as amplification for sequencing. Examples of polymerases with 3' to 5' exonuclease activity include: KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1; Vent, which is extracted from Thermococcus litoralis; Pfu DNA polymerase, which is extracted from Pyrococcus furiosus; and Pwo, which is extracted from Pyrococcus woesii.

Size and other limitations

PCR works readily with DNA of lengths two to three thousand basepairs, but above this length the polymerase tends to fall off, and the typical heating cycle does not leave enough time for polymerisation to complete. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These special polymerases are often polymerases fused to a processivity-enhancing DNA-binding protein, making them literally "stick" to the DNA longer[4][5].

Non specific priming

The non specific binding of primers is always a possibility due to sequence duplications, non-specific binding and partial primer binding, leaving the 5' end unattached. This is increased by the use of degenerate sequences or bases in the primer. Manipulation of annealing temperature and magnesium ion (which stabilise DNA and RNA interactions) concentrations can increase specificity. Non-specific priming can be prevented during the low temperatures of reaction preparation by use of "hot-start" polymerase enzymes where the active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to 95˚C during the denaturation step of the first cycle.

Practical modifications to the PCR technique

Nested PCR - Nested PCR is intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. Two sets of primers are used in two successive PCR runs, the second set intended to amplify a secondary target within the first run product. This is very successful, but requires more detailed knowledge of the sequences involved.

Inverse PCR - Inverse PCR is a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of digestions and self ligation before cutting by an endonuclease, resulting in known sequences at either end of the unknown sequence.

RT-PCR - RT-PCR (Reverse Transcription PCR) is the method used to amplify, isolate or identify a known sequence from a cell or tissues RNA library. Essentially normal PCR preceded by Reverse transcriptase (to convert the RNA to cDNA) this is widely used in expression mapping, determining when and where certain genes are expressed.

Assembly PCR - Assembly PCR is the completely artificial synthesis of long gene products by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments serve to order the PCR fragments so that they selectively produce their final product.

Asymmetric PCR - Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.

Quantitative PCR - Q-PCR (Quantitative PCR) is used to rapidly measure the quantity of PCR product (preferably real-time), thus is an indirect method for quantitatively measuring starting amounts of DNA, cDNA or RNA. This is commonly used for the purpose of determining whether a sequence is present or not, and if it is present the number of copies in the sample. There are 3 main methods which vary in difficulty and detail. Quantitative real-time PCR is often confusingly known as RT-PCR (Real Time PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR can also refer to reverse transcription PCR, which even more confusingly, is often used in conjunction with Q-PCR. This method uses fluorescent dyes and probes to measure the amount of amplified product in real time.

Touchdown PCR - Touchdown PCR is a variant of PCR that reduces nonspecific primer annealing by more gradually lowering the annealing temperature between cycles. As higher temperatures give greater specificity for primer binding, primers anneal first as the temperature passes through the zone of greatest specificity.

Hot-start PCR is a technique that reduces non-specific priming that occurs during the preparation of the reaction components. The technique may be performed manually by simply heating the reaction components briefly at the melting temperature before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Colony PCR - Bacterial clones (E.coli) can be screened for the correct ligation products. Selected colonies are picked with a sterile toothpick from an agarose plate and dabbed into the master mix or sterile water. Primers (and the master mix) are added - the PCR protocol has to be started with an extended time at 95oC when standard polymerase is used or with shorten denaturation step at 100oC and special chimeric DNA polymerase[9]. RACE-PCR - Rapid amplification of cDNA ends.

Multiplex-PCR - The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be elicited from a single test run that otherwise would require several times the reagents and technician time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction and amplicon sizes should be separated by enough difference in final base pair length to form distinct bands via gel electrophoresis.

Methylation Specific PCR - Methylation Specific PCR (MSP) is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCR reactions are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

History

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 1 one night in his car[2]. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in Chemistry with Michael Smith in 1993. As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat." DNA polymerase occurs naturally in living organisms. In cells it functions to duplicate DNA when cells divide in mitosis and meiosis. Polymerase works by binding to a single DNA strand and creating the complementary strand. In the first of many original processes, the enzyme was used in vitro (in a controlled environment outside an organism). The double-stranded DNA was separated into two single strands by heating it to 94°C (201°F). At this temperature, however, the DNA polymerase used at the time were destroyed, so the enzyme had to be replenished after the heating stage of each cycle. The original procedure was very inefficient, since it required a great deal of time, large amounts of DNA polymerase, and continual attention throughout the process. Later, this original PCR process was greatly improved by the use of DNA polymerase taken from thermophilic bacteria grown in geysers at a temperature of over 110°C (230°F). The DNA polymerase taken from these organisms is stable at high temperatures and, when used in PCR, does not break down when the mixture was heated to separate the DNA strands. Since there was no longer a need to add new DNA polymerase for each cycle, the process of copying a given DNA strand could be simplified and automated. One of the first thermostable DNA polymerases was obtained from Thermus aquaticus and was called "Taq." Taq polymerase is widely used in current PCR practice. A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations (errors) in the DNA sequence, since it lacks 3'→5' proofreading exonuclease activity. Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied DNA sequence. However these enzymes polymerise DNA at a much slower rate than Taq. Combinations of both Taq and Pfu are available nowadays that provide both high processivity (fast polymerisation) and high fidelity (accurate duplication of DNA). PCR has been performed on DNA larger than 10 kilobases, but the average PCR is only several hundred to a few thousand bases of DNA. The problem with long PCR is that there is a balance between accuracy and processivity of the enzyme. Usually, the longer the fragment, the greater the probability of errors.

Patent wars

The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme is also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected. A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. Interestingly, it seems possible that the legal arguments will extend beyond the life of the original PCR and Taq polymerase patents, which expired on March 28, 2005.[12]

 

References

  1. The history of PCR: Smithsonian Institution Archives, Institutional History Division. Retrieved 24 June 2006.
  2. a b Mullis, Kary (1998). Dancing Naked in the Mind Field. New York: Pantheon Books. ISBN 0-679-44255-3. 
  3. Sambrook, Joseph; and David W. Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-87969-576-5. 
  4. Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI (2002). "Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases". Proc Natl Acad Sci. 99: 3510-13515. PMID 12368475.
  5. Demidov VV (2002). "A happy marriage: advancing DNA polymerases with DNA topoisomerase supplements". Trends Biotechnol. 20: 491. DOI:10.1016/S0167-7799(02)02101-7.
  6. Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications". Trends Biotechnol. 22: 253-260. PMID 15109812.
  7. Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2004). Thermostable Chimeric DNA Polymerases with High Resistance to Inhibitors”, DNA Amplification: Current Technologies and Applications. Horizon Bioscience, pp. 3-20. ISBN 0-9545232-9-6. 
  8. Forterre P (2006). "DNA topoisomerase V: a new fold of mysterious origin". Trends Biotechnol. 24: 245-247. PMID 16650908.
  9. a b Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes, Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0. 
  10. Taneja B, Patel A, Slesarev A, Mondragon A (2006). "Structure of the N-terminal fragment of topoisomerase V reveals a new family of topoisomerases". EMBO J. 25: 398-408. PMID 16395333.
  11. Rabinow, Paul (1996). Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press. ISBN 0-226-70146-8. 
  12. Advice on How to Survive the Taq Wars ¶2: GEN Genetic Engineering News Biobusiness Channel: Article. May 1 2006 (Vol. 26, No. 9).

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