Polymerase Chain Reaction (PCR) - Variations to the System

45 min Read

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.

abm offers a comprehensive collection of DNA polymerases and PCR MasterMixes suitable for different PCR variations.


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:

  • Single template PCR reactions: this uses a single template and several pairs of primers to amplify several regions within a template
  • Multiple template PCR reactions: where multiple templates and primers can be present in the same reaction tube

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:

  • Internal Controls: False negatives due to reaction failure are often showed in multiplex assays because each amplicon provides an internal control for the other amplicons.
  • Efficiency: It saves preparation time, as well as cost of polymerases and templates (2)(3)(4).
  • 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).

    Multiplex PCR

    Figure 1 – Multiplex PCR


    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).

    Nested PCR

    Figure 2 – Nested PCR

    Asymmetric PCR

    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).

    Asymmetric PCR

    Figure 3 – Asymmetric PCR

    Assembly PCR

    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).

    Assembly PCR

    Figure 4 – Assembly PCR

    Touchdown PCR

    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:

    • Phase 1 of the touchdown PCR programming uses an annealing temperature that is approximately 10°C above the calculated Tm (melting temperature). In this phase, the temperature is reduced by 1°C every successive cycle until the calculated Tm range is reached. This is done for a total of 10-15 cycles.
    • Phase 2 follows generic PCR amplification for up to 20-25 cycles using the final annealing temperature reached in the touchdown phase. The cycles and temperature drops during the touchdown phase can be adjusted to 1-3 cycles per 1-3°C drop in temperature if non-specific products are still observed or if the yield is low (13).

    Touchdown PCR

    Figure 5 – Touchdown PCR

    Digital PCR

    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).

    Digital PCR

    Figure 6 – Digital PCR

    Suicide PCR

    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).

    Site Directed Mutagenesis

    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).

    Site directed mutagenesis

    Figure 7 – Site Directed Mutagenesis


    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:

    • Denaturation stage: DNA is denatured at a high temperature – usually 95 °C
    • Intermediate annealing stage: set an intermediate annealing temperature that allows hybridization of mutant and wildtype allele DNA to one another. Because the mutant allele DNA forms the minority of DNA in the mixture they will be more likely to form mismatch heteroduplex DNA with the wildtype DNA
    • Melting stage: these heteroduplexes will more readily melt at lower temperatures so they are selectively denatured at the Tc while the homo-duplex will not melt and remain double stranded
    • Primer annealing stage: the homo-duplex DNA will preferentially remain double stranded and not be available for primer annealing
    • Extension stage: the DNA polymerase will extend complementary to the template DNA. Since the heteroduplex DNA is used as template, a larger proportion of minor variant DNA will be amplified and be available for subsequent rounds of PCR.


    Figure 8 – Cold-PCR

    Isothermal PCR

    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).

    • Williams DL, Gillis TP, Booth RJ, Looker D and Watson JD. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. The Journal of infectious diseases. 1990; 162(1):193-200.
    • Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN and Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic acids research. 1988; 16(23):11141-11156.
    • Ballabio A, Ranier JE, Chamberlain JS, Zollo M and Caskey CT. Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification. Human genetics. 1990; 84(6):571-573.
    • Hayden MJ, Nguyen TM, Waterman A and Chalmers KJ. Multiplex-ready PCR: a new method for multiplexed SSR and SNP genotyping. BMC genomics. 2008; 9:80.
    • Wen DX and Zhang CQ. Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments. Plant Methods. 2012; 8.
    • Haff LA. Improved quantitative PCR using nested primers. PCR methods and applications. 1994; 3(6):332-337.
    • Hurtado A, Aduriz G, Moreno B, Barandika J and Garcia-Perez AL. Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes. Veterinary parasitology. 2001; 102(1-2):17-27.
    • Sanchez JA, Pierce KE, Rice JE and Wangh LJ. Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proceedings of the National Academy of Sciences of the United States of America. 2004; 101(7):1933-1938.
    • Pierce KE and Wangh LJ. Linear-after-the-exponential polymerase chain reaction and allied technologies. Real-time detection strategies for rapid, reliable diagnosis from single cells. Methods in molecular medicine. 2007; 132:65-85.
    • Stemmer WP, Crameri A, Ha KD, Brennan TM and Heyneker HL. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995; 164(1):49-53.
    • Kosuri S, Eroshenko N, Leproust EM, Super M, Way J, Li JB and Church GM. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature biotechnology. 2010; 28(12):1295-1299.
    • Don RH, Cox PT, Wainwright BJ, Baker K and Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic acids research. 1991; 19(14):4008.
    • Korbie DJ and Mattick JS. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature protocols. 2008; 3(9):1452-1456.
    • Pohl G and Shih LM. Principle and applications of digital PCR. Expert Rev Mol Diagn. 2004; 4(1):41-47.
    • Fournier PE and Raoult D. Suicide PCR on skin biopsy specimens for diagnosis of rickettsioses. Journal of clinical microbiology. 2004; 42(8):3428-3434.
    • Raoult D, Aboudharam G, Crubezy E, Larrouy G, Ludes B and Drancourt M. Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death. Proceedings of the National Academy of Sciences of the United States of America. 2000; 97(23):12800-12803.
    • Moller A and Brinkmann B. Pcr-Vntrs (Pcr-Variable Number of Tandem Repeats) in Forensic-Science. Cell Mol Biol. 1995; 41(5):715-724.
    • Zajaczek S, Gorski B, Debniak T, Podolski J, Lubinski J, Krzystolik Z, Iwanicka T and Sagan Z. [VNTR-PCR in diagnosis of inherited Rb gene mutation]. Klinika oczna. 1994; 96(8-9):290-292.
    • McGregor D, Galvin P, Sadusky T and Cross T. PCR amplification of a polymorphic minisatellite VNTR locus in whiting (Merlangius merlangus L.). Animal genetics. 1996; 27(1):49-51.
    • Batanian JR, Ledbetter DH and Fenwick RG. A simple VNTR-PCR method for detecting maternal cell contamination in prenatal diagnosis. Genetic testing. 1998; 2(4):347-350.
    • Edelheit O, Hanukoglu A and Hanukoglu I. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC biotechnology. 2009; 9:61.
    • Milbury CA, Li J, Liu PF and Makrigiorgos GM. COLD-PCR: improving the sensitivity of molecular diagnostics assays. Expert Rev Mol Diagn. 2011; 11(2):159-169.
    • Li J and Makrigiorgos GM. COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing. Biochem Soc T. 2009; 37:427-432.
    • Yan L, Zhou J, Zheng Y, Gamson AS, Roembke BT, Nakayama S and Sintim HO. Isothermal amplified detection of DNA and RNA. Molecular bioSystems. 2014; 10(5):970-1003.