Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism (such as yeast). Like amplification using living organisms, the technique allows a small amount of the DNA molecule to be amplified exponentially. However, because it 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 is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments, usually up to 10 kb. Certain methods can copy fragments up to 47 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.

PCR, as currently practiced, requires several basic components:

• 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
• Deoxynucleotides-triphosphate, from which the DNA Polymerase builds the new DNA
• Buffer, which provides a suitable chemical environment for the DNA Polymerase

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. To prevent evaporation of the reaction mixture (typically volumes between 15-100 µl per tube), a heated lid is placed on top of the reaction tubes or a layer of oil is put on the surface of the reaction mixture.

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 (T_m) 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. The melting temperature increases with the length of the primer. 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 temperature required to melt it. 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.

Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine might be "ATH" [A = adenine, T = thymine, H = adenine, thymine, or cytosine]. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. This problem can be partly solved by using touchdown PCR.

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

• GC-content should be between 40-60%.
• Calculated T_m for both primers used in reaction should not differ >5°C and T_m of the amplification product should not differ from primers by >10°C.
• Annealing temperature usually is 5°C below the calculated lower T_m. 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 template.

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. Also certain polymerases are activated at this step (see hot-start PCR).

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 (45-60°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: 1-2 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. 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. This differs from all other elongation steps, only in that it is longer, typically 10-15 minutes. This last step is highly recommendable if the PCR product is to be ligated into a T vector using TA-cloning.

The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

Example

The times and temperatures given in this example are taken from a PCR program that was successfully used on a 250 bp fragment of the C-terminus of the insulin-like growth factor (IGF). 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.

The PCR Process

The PCR process consists of the following steps:

1. Initialization. The mixture is heated at 96°C for 5 minutes to ensure that the DNA strands as well as the primers have melted. The DNA Polymerase can be present at initialization, or it can be added after this step.
2. Melting, where it is heated at 96°C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.
3. Annealing by heating at 68°C for 30 seconds. The primers are jiggling around, caused by the Brownian motion. Short bondings are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the Tm of the double-stranded region between the template and the primer is greater than the annealing or extension temperature.
4. Elongation by heating 72°C for 45 seconds. This is the ideal working temperature for the polymerase. The primers, having been extended for a few bases, already have a stronger hydrogen bond to the template than the forces breaking these attractions. Primers that are on positions with no exact match, melt away from the template (because of the higher temperature) and are not extended.

The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

1. Steps 2-4 are repeated 25 times. With good primers and fresh polymerase, 15 to 20 cycles is sufficient.
2. Mixture is held at 7°C. This is useful if one starts the PCR in the evening just before leaving the lab, so it can run overnight. The DNA will not be damaged at 7°C after just one night.

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.

Optimization

Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in lab environment (bacteria, viruses, own DNA 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.

Practical Modifications

• 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 transcription by Reverse transcriptase (to convert the RNA to cDNA) this is widely used in expression mapping, determining when and where certain genes are expressed.

• 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.

• 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 95^^C.

• 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 illicited 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.

Recent Developments

A more recent method which excludes a temperature cycle, but uses enzymes, is helicase-dependent amplification. Other recent developments include TAIL-PCR (Liu et al. 1995) or thermal asymmetric interlaced PCR, and also Meta-PCR (Andrew Wallace) which allows one to optimize amplification and direct sequence analysis of complex genes.