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The polymerase chain reaction (PCR) is a revolutionary method developed by Kary Mullis in the 1980s [1] and is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cDNA template can be amplified from small amounts to many thousand- to a million-fold using sequence specific primers, heat stable DNA polymerases, and thermal cycling.
PCR is a highly versatile technique and has been modified in different ways to suit specific applications. This section will summarize some of the different types of PCR, including their working principles, their applications, their advantages, and their disadvantages.
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Multiplex PCR is a widely used molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplex-PCR assay, different target DNA sequences can be amplified simultaneously by using multiple primer pairs in a reaction mixture. Annealing temperature and primer sets should be optimized so that all primer pairs can work correctly within a single reaction. Amplicon sizes of different genes such as their base pair length should be different so that distinct bands can be visualized by gel electrophoresis. Otherwise, distinct amplicons should be differentiated and visualized using primers dyed with different colour fluorescent dyes. Multiplex PCR can be broadly divided into:
Multiplex PCR has been used in pathogen identification, high throughput SNP genotyping, mutation analysis, gene deletion analysis, template quantification, linkage analysis, RNA detection and forensic studies. The advantages of Multiplex PCR include:
Nevertheless, the Multiplex PCR method has several disadvantages, including a complex system with many primers, a low amplification efficiency and efficiency variation on different templates (5).
Nested PCR is used to increase the specificity of DNA amplification by reducing the non-specific amplification of DNA. A nested PCR assay has 2 sets of primers (an outer pair and an inner pair) for a single locus and two successive PCRs. In the first PCR run, the outer pair of primers are used to generate DNA products similar to regular PCRs and thus their DNA products may contain amplification of non-specific DNA fragments. These products then enter a second PCR run that uses the second set “inner” primers whose binding sites are located after the 3'dn of the outer primer pair and either completely or partially different from the outer primer pair used in the first PCR reaction. Therefore a second, shorter PCR product will be produced after the products from the first run. If the wrong locus was amplified by mistake in the first run, it’s very unlikely it would also be amplified a second time by the second pair of primers. In this way, nested PCR greatly increases the specificity of PCR. Nested primers are used as important controls for many experiments involving unknown genome sequences (6) such as when amplifying a particular member of a polymorphic gene family or amplifying from a clinical specimen containing a heterogeneous population of sample inputs. A drawback with this technique is that addition of a second pair of primers after the first PCR run increases the risk of nonspecific contamination (7).
In an asymmetric PCR, the reaction preferentially amplifies one DNA strand in a double-stranded DNA template. Thus it is useful when amplification of only one of the two complementary strands is needed such as in sequencing and hybridization probing. The whole PCR process is similar to regular PCR, except that the amount of primer for the targeted strand is much more than that of the non-targeted strand. As the asymmetric PCR progresses, the lower concentrated limiting primer is quantitatively incorporated into newly synthesized double stranded DNA and used up. Consequently, linear synthesis of the targeted single DNA strand from the excess primer are formed after depletion of the limiting primer.
Asymmetric PCR is not widely used because it has low reaction efficiency and it is hard to optimize the proper primer ratios, the amounts of starting material, and the number of amplification cycles. Limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature (8). Recently, this process has been renamed as Linear-After-The –Exponential-PCR (LATE-PCR) where the limiting lower concentrated primer has a higher melting temperature than the more highly concentrated primer to maintain reaction efficiency (9).
Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of seed oligonucleotides with short complimentary segments. The seed oligonucleotides are designed to be either part of the sense or antisense strand of the target DNA. The complimentary segments determine the order of the seed oligonucleotides, thereby selectively producing the final long DNA product. During the polymerase cycles, the oligonucleotides anneal to complementary fragments and then are filled in by the DNA polymerase. After the initial construction of the long DNA sequence, primers to both ends of the long DNA sequence are added and a regular PCR reaction is performed. The complete target sequence is then isolated by gel purification. A typical reaction consists of oligonucleotides that are ~50 base pairs long each overlapping by about 20 base pairs. The reaction with all the oligonucleotides is then carried out for ~30 cycles followed by an additional 23 cycles with the end primers (10). Assembly PCR is a very flexible technique for producing novel gene sequences because single-stranded oligos or a mix of single- and double-stranded DNA can be used to produce longer genes of up to several thousand base pairs (11).
In “Touch Down” PCR, the annealing temperature of the earlier PCR cycles is set to just below the melting temperature of the primer sets. The annealing temperature is then incrementally reduced for every subsequent set of cycles (associated parameters can be chosen by the experimenter). The principle behind this is that the annealing temperature during a PCR reaction determines the specificity of primer annealing. At high annealing temperatures, only very specific base pairing between the primer and the template will occur and thus the first sequence amplified is the one between the regions of greatest primer specificity and the one of interest. Then the amplified fragments will be further amplified during subsequent lower temperature runs with more efficiency. In this way, the number of amplified targeted sequences would be in excess compared to the number of amplified non-specific sequences. Touch Down PCR increases the specificity of PCR by using higher annealing temperatures at the earlier cycles and increases the efficiency by lowering the annealing temperatures gradually toward the end of the cycles. This method dramatically increases the quality and outcome of PCR (12). A typical Touch Down PCR cycling condition has two phases:
Digital PCR is a novel approach for the absolute quantification of nucleic acid. In Digital PCR, a sample of DNA or cDNA is separated into a large number of partitions/wells and PCR reactions are carried out in each partition individually. Some of the wells contain the target molecule (positive) and thus have positive PCR reactions while others do not (negative) and have negative PCR reactions. PCR can amplify a single DNA template a million-fold or more. The amplicons are then hybridized with fluorescent probes. When there is no targeted molecule in that well, no signal accumulates. At the end, the proportions of positive and negative signals are used to generate an absolute number of targeted molecules in the sample. Digital PCR has several advantages. It doesn’t need references or endogenous controls. It has much higher accuracy and sensitivity due to more PCR replicates. Therefore, it’s widely used for analysis of copy number alterations, rare mutations, next-generation sequencing, etc (14).
Suicide PCR avoids false positive amplifications as its top priority. Therefore it is typically used in paleogenetics (the study of the past through the examination of preserved genetic material from the remains of ancient organisms) or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is of the highest priority. For example, suicide PCR was utilized to improve the diagnosis of rickettsioses on eschar biopsy specimens taken prior to antibiotic therapy (15). Suicide PCR was originally described in a study to verify the presence of the microbe Yersinia pestis in dental samples obtained from 14th Century graves of people supposedly killed by plague during the medieval Black Death epidemic (16). In this method, target-specific primer pairs can be used only one time and should never be used in any positive control PCR reaction. Multiple sets of primers can be tested until an amplicon of the expected size is yielded. This amplicon is then sequenced to confirm its identity. To ensure no DNA from previous PCR reactions can contaminate the current PCR assay and generate false positive controls (16), it is important that the targeted sequence has never previously been amplified in the same lab.
Variable number tandem repeat (VNTR) is a region where short nucleotide sequences occur as tandem repeats. It can be found on many chromosomes and demonstrates variations in lengths between individuals. Thus VNTR can be used for parental and personal identification. VNTR PCR targets the VNTR region. Analysis of the sample's genotypes is usually accomplished through sizing of the amplified products by gel electrophoresis. This technique is used in genetics, biology research, forensics(17) and DNA fingerprinting. In paritcular, analysis of smaller VNTR segments know as short tandem repeats is the basis for DNA fingerprinting databases (18)(19)(20).
This is an important molecular biology method that introduces specific and intentional mutations to a DNA sequence of a gene or any genetic products. During this process, a short DNA primer is synthesize which contains the expected mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA sequence of interest. The mutation can be a single or multiple base change, deletion or insertion. The primer is then elongated by DNA polymerase. The amplified gene contains the mutated site, which is then incorporated into a host cell as a vector and cloned. Finally, mutants are selected by DNA sequencing to select those with desired mutations. This method can be used to study the function of a gene or protein, or for creating variants of an enzyme with new and improved functions (21).
COLD-PCR (co-amplification at lower denaturation temperature-PCR) differs from the traditional PCR protocol in that it can preferentially amplify and identify minority alleles and low-level somatic DNA mutations from a mixture of wildtype and mutation-containing DNA. It is therefore useful for the detection of mutations and is particularly important for early cancer detection from tissue biopsies and body fluids, monitoring of therapy outcome and cancer remission or relapse, assessment of residual disease after surgery or chemotherapy, and molecular profiling for prognosis or tailoring therapy to individual patients (22) (23).
The underlying principle of COLD-PCR is that single nucleotide mismatches will slightly lower the melting temperature (Tm) of the double-stranded DNA, to an extent that depends on the sequence context and position of the mismatch. Just below the Tm there is a critical denaturation temperature (Tc) wherein PCR efficiency drops abruptly as a result of the limited number of denatured amplicons. This difference in PCR efficiency, at specifically defined denaturation temperatures, can be used to selectively enrich minority (or low-abundance mutant) alleles throughout the course of PCR.
A typical COLD-PCR cycle includes:
Unlike conventional PCR which needs a thermocycling machine to separate two DNA strands and to amplify the required fragment, isothermal PCR can amplify DNA in isothermal conditions without the need of a thermocycling apparatus. DNA polymerase replicates DNA with the aid of various accessory proteins. Recent identification of these proteins has enabled development of new in vitroisothermal DNA amplification methods, mimicking the in vivo mechanisms. There are several types of isothermal nucleic acid amplification methods such as nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification. These isothermal amplification protocols have various advantages, including their extreme speed and their independence from thermocyclers. Therefore, isothermal PCR is suitable for clinical diagnostics and biosafety examinations (24).
