36 Types of PCR (Polymerase Chain Reaction) with Definitions and Applications

Amplified fragment length polymorphism (AFLP) PCR
Allele-specific PCR
Alu PCR
Assembly PCR
Asymmetric PCR
COLD PCR
Colony PCR
Conventional PCR
Digital PCR (dPCR)
Fast cycling PCR
High Fidelity PCR
High-Resolution Melt (HRM) PCR
Hot start PCR
In-situ PCR
Intersequence specific (ISS) PCR
Inverse PCR
LATE (Linear-After-The-Exponential) PCR
Ligation mediated PCR
Long-Range PCR
Methylation-specific PCR (MSP)
Miniprimer PCR
Multiplex PCR
Nanoparticle-Assisted PCR (nanoPCR)
Nested PCR
Overlap extension PCR (OE-PCR)
Real-Time PCR (Quantitative PCR (qPCR))
Repetitive sequence-based PCR
Reverse Transcriptase PCR (RT-PCR)
Reverse-Transcriptase Real-Time PCR (RT-qPCR)
RNase H-dependent PCR
Single Specific Primer-PCR (SSP-PCR)
Solid Phase PCR
Suicide PCR
Thermal asymmetric interlaced PCR (TAIL-PCR)
Touch down PCR
Variable Number of Tandem Repeats (VNTR) PCR

References and Sources

1. Amplified fragment length polymorphism (AFLP) PCR

Amplified Fragment Length Polymorphism (AFLP) PCR is a PCR-based technique that relies on the selective amplification of digested DNA fragments to create unique genetic fingerprints for the genome being studied.
It is capable of rapidly generating a large number of marker fragments for any organism, even when there is no prior information available about its genomic sequence.
The method begins with the digestion of genomic DNA using restriction enzymes, followed by the ligation of adaptors to the sticky ends of the resulting DNA fragments.
A specific subset of these restriction fragments is then chosen for amplification using primers that match the adaptor sequences.
The amplified DNA fragments are subsequently separated and visualized through denaturing agarose gel electrophoresis.
AFLP PCR has a wide range of applications, including assessing genetic diversity within or among closely related species, inferring population-level phylogenies and biogeographic patterns, generating genetic linkage maps, and determining genetic relatedness among different cultivars.

2. Allele-specific PCR

Allele-specific PCR (AS-PCR) is a polymerase chain reaction technique that uses allele-specific primers to detect single nucleotide polymorphisms (SNPs).
This method is also known as the amplification refractory mutation system (ARMS-PCR), referring to the use of two separate primers designed to target two different alleles.
One primer set is specific for the mutant allele and does not support amplification of the normal allele, while the other primer set is specific for the normal allele and is refractory to amplification of the mutant version.
The primers are designed with modified 3’ ends so that one set selectively amplifies the normal allele, whereas the other selectively amplifies the mutant allele.
This intentional mismatch at the primer’s 3’ end enables amplification of only one specific allele at a time.
AS-PCR is extensively used for detecting single-gene point mutations, including those responsible for diseases such as sickle cell anemia and thalassemia.
The technique is also applied in directly determining ABO blood group genotypes.

3. Alu PCR

Alu PCR is a fast and simple DNA fingerprinting method that examines multiple genomic regions flanked by Alu repetitive elements simultaneously.
Alu elements are short DNA sequences originally identified through the activity of the Arthrobacter luteus (Alu) restriction endonuclease.
These elements are among the most abundant transposable elements in the human genome, contribute to genome evolution, and are widely used as genetic markers.
In Alu PCR, two fluorochrome-labeled primers designed to match Alu sequences are used for amplification, and the resulting PCR products are subsequently analyzed.
Alu insertions have been associated with numerous inherited human disorders and several types of cancer; therefore, this technique is valuable for detecting such diseases and related genetic mutations.

4. Assembly PCR

Assembly PCR is a technique used to construct large DNA sequences by assembling multiple shorter oligonucleotide fragments.
While conventional PCR typically uses oligonucleotides around 18 base pairs in length, assembly PCR employs longer oligos up to 50 base pairs, to ensure accurate hybridization.
Throughout the PCR cycles, these oligonucleotides anneal to their complementary overlapping fragments, and DNA polymerase extends them to create longer sequences.
With each cycle, fragment length increases in a somewhat random pattern, depending on which complementary oligonucleotides successfully anneal to one another.
Assembly PCR is commonly used to enhance the yield of target proteins and is also useful for generating large quantities of RNA required for structural or biochemical research.

5. Asymmetric PCR

Asymmetric PCR is a modified form of PCR designed to preferentially amplify only one strand of the target DNA rather than both.
Unlike standard PCR, it uses a large excess of one primer (for the strand of interest) and a limiting amount of the opposite primer.
As the reaction proceeds, the limiting primer becomes fully consumed, producing double-stranded DNA only during the early cycles.
Once the limiting primer is depleted, amplification continues using only the excess primer, resulting in linear synthesis of single-stranded DNA.
This method is particularly useful when single-stranded DNA is required, such as for DNA sequencing, probe generation, and hybridization assays.

6. COLD PCR

Co-amplification at Lower Denaturation Temperature PCR (COLD-PCR) is an advanced PCR technique designed to selectively enrich low-abundance DNA variants within a mixture of wild-type and mutant sequences, regardless of the mutation type or its position within the amplicon.

The method relies on modifying the critical denaturation temperature, allowing mutation-containing DNA to melt preferentially over wild-type DNA.
After denaturation, an intermediate annealing step promotes the formation of heteroduplexes (wild-type/mutant hybrids). These mismatched duplexes have slightly altered melting temperatures.
Because heteroduplexes melt more easily, they become the preferred templates for amplification, leading to selective enrichment of rare mutant alleles over successive cycles.
COLD PCR is particularly valuable in detecting low-frequency mutations in oncology samples, especially in heterogeneous tumors and bodily fluids.
It is also widely applied in minimal residual disease (MRD) detection, post-treatment monitoring, disease staging, and molecular profiling, enabling more personalized therapeutic decisions.

7. Colony PCR

Colony PCR is a rapid screening technique used to identify whether a bacterial (or yeast) colony contains a plasmid with the desired DNA insert. The method relies on primers designed specifically for the inserted DNA sequence.

In this technique, bacterial colonies are directly used as the DNA template, without the need for plasmid extraction.
Two sets of primers are typically employed:

Insert-specific primers that amplify the target DNA insert.
Vector-specific flanking primers that amplify regions of the plasmid outside the insertion site.

A small amount of a bacterial colony is picked and added directly into the PCR master mix containing all required reagents.
Colony PCR is primarily used to confirm successful ligation and insertion of DNA into plasmids during cloning procedures in bacteria and yeast.

8. Conventional PCR

Conventional Polymerase Chain Reaction (PCR) is an in-vitro technique that replicates DNA, allowing a specific “target” sequence to be amplified millions of times within a few hours.

PCR synthesizes specific DNA fragments using DNA polymerase, an enzyme responsible for replicating genetic material.
The process begins when short DNA fragments called primers bind to complementary sequences on the target DNA, defining the region to be amplified.
DNA polymerase then extends these primers, generating new complementary strands. Through repeated cycles of denaturation, annealing, and extension, billions of copies of the desired DNA segment are produced.
Conventional PCR is widely used for selective DNA isolation, DNA amplification, qualitative detection, and quantification in medical diagnostics, infectious disease detection, forensic investigations, and various research applications.

9. Digital PCR (dPCR)

Digital PCR (dPCR) is an advanced quantitative PCR technique that enables highly sensitive and precise measurement of DNA or RNA molecules in a sample.

In dPCR, the reaction mixture is partitioned into thousands to millions of tiny wells or droplets before amplification, so each partition contains either zero or one copy of the target sequence.
After PCR amplification, partitions are analyzed for fluorescence signals:

Fluorescent wells are counted as positive (1).
Non-fluorescent wells are negative (0).

By assessing the number of positive and negative reactions, the absolute quantity of the target nucleic acid in the original sample is calculated using Poisson statistics.
dPCR provides high precision, does not require a standard curve, and is ideal for detecting low-level variants, rare mutations, and small-fold changes.
It is widely used to quantify DNA/RNA viruses, bacteria, and parasites in diverse clinical specimens—especially when reliable calibration standards are unavailable.

10. Fast cycling PCR

Fast cycling PCR is a modified PCR technique designed to amplify target DNA sequences in a significantly shorter time compared to conventional PCR.

The fundamental principles remain the same as standard PCR; however, the reaction time per cycle is greatly reduced.
Specialized fast-cycling buffers enhance the binding affinity of Taq DNA polymerase to short single-stranded DNA regions, allowing primer annealing to occur in as little as 5 seconds.
As a result, complete PCR amplification can be achieved in a fraction of the usual time.
Fast cycling PCR is highly valuable in workflows that require rapid turnaround, such as quick mutation screening, urgent diagnostic assays, and time-sensitive molecular biology applications.

11. High Fidelity PCR

High-fidelity PCR is a modified PCR technique that uses DNA polymerases with exceptionally low error rates, ensuring highly accurate replication of the target DNA sequence.

These specialized polymerases possess a strong binding affinity for the correct nucleoside triphosphates, which enhances the accuracy of nucleotide incorporation during amplification.
If an incorrect nucleotide enters the polymerase active site, the structural arrangement of the enzyme slows down incorporation, reducing the likelihood of errors.
Many high-fidelity polymerases also contain 3′→5′ exonuclease proofreading activity, which removes misincorporated bases and further improves accuracy.
High-fidelity amplification is crucial for applications that require precise DNA sequence integrity, such as cloning, site-directed mutagenesis, single nucleotide polymorphism (SNP) analysis, and next-generation sequencing (NGS) library preparation.

12. High-Resolution Melt (HRM) PCR

High-Resolution Melt (HRM) PCR is a highly sensitive technique used for detecting mutations, polymorphisms, and epigenetic variations in double-stranded DNA samples.

HRM is significantly more cost-effective compared to other genotyping methods such as DNA sequencing and TaqMan SNP assays, making it ideal for large-scale genotyping studies.
It is fast and efficient, allowing accurate genotyping of large numbers of samples in a short time.
The technique is simple and user-friendly. With a well-designed HRM assay, powerful genotyping can be performed even by non-geneticists, provided they have access to an HRM-capable real-time PCR instrument.
HRM works by precisely monitoring the melting behavior of PCR-amplified DNA. Small sequence differences cause detectable changes in the melt curve, enabling discrimination between variants.

13. Hot start PCR

Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) designed to minimize the formation of undesired products and primer-dimers caused by non-specific DNA amplification at room temperature.
The fundamental principle of hot start PCR involves keeping one or more key reagents separate from the reaction mixture until the mixture reaches the denaturation temperature during the initial heating step.
This approach significantly reduces non-specific binding and primer-dimer formation, often resulting in higher product yields.
Hot start PCR also requires less manual intervention and lowers the risk of contamination during the reaction setup.

14. In-situ PCR

In-situ PCR (In-Situ Polymerase Chain Reaction) is a technique used to detect very small amounts of rare nucleic acid sequences within frozen or paraffin-embedded cells or tissue sections, allowing localization of these sequences within the cells.
The method begins with tissue fixation, which preserves cell morphology, followed by treatment with proteolytic enzymes to allow PCR reagents access to the target DNA.
The target DNA sequences are then amplified in situ by the PCR reagents and subsequently detected using standard immunocytochemical protocols.
In-situ PCR is useful for diagnosing infectious diseases, quantifying DNA, detecting very low amounts of DNA, and is widely employed in studies of organogenesis and embryogenesis.

15. Intersequence specific (ISS) PCR

Intersequence-Specific PCR (ISSR-PCR) is a DNA fingerprinting technique that employs primers designed from specific repetitive segments distributed throughout the genome to generate a unique genetic fingerprint.
The method uses microsatellites, typically 16–25 base pairs long, as primers in a single-primer PCR reaction that targets multiple genomic loci, primarily amplifying the inter-SSR sequences of varying lengths.
ISSR-PCR is applied in genomic fingerprinting, assessing genetic diversity, phylogenetic analysis, genome mapping, and gene tagging.

16. Inverse PCR

Inverse PCR is a variant of the polymerase chain reaction designed to amplify DNA when only a single sequence of the target is known.
Unlike conventional PCR, which requires primers complementary to both ends of the target DNA, inverse PCR can amplify DNA using primers designed from just one known sequence.
The technique involves restriction digestion of the DNA followed by ligation, producing a looped fragment that allows PCR priming from the known sequence.
The looped DNA is then amplified using a temperature-sensitive DNA polymerase, similar to standard PCR.
Inverse PCR is particularly useful for identifying the insertion sites of transposons and retroviruses within host DNA.

17. LATE (Linear-After-The-Exponential) PCR

LATE (Linear-After-The-Exponential) PCR is a modification of asymmetric PCR that employs a limiting primer with a higher melting temperature than the excess primer, ensuring that reaction efficiency is maintained even as the limiting primer concentration declines during the reaction.
The process starts with an exponential amplification phase, similar to conventional PCR, where both primers contribute to efficient DNA synthesis.
Once the limiting primer is exhausted, the reaction transitions to a linear amplification phase, during which single-stranded DNA is continuously produced over many additional thermal cycles.

18. Ligation mediated PCR

Ligation-mediated PCR is a variant of conventional PCR that allows amplification when only one end of the target DNA is initially known, with the second end added via ligation of a unique DNA linker.
The technique uses small DNA fragments called “linkers” or adaptors, which are ligated to the target DNA fragments.
PCR primers complementary to the linker sequences are then used to amplify the target DNA fragments.
Ligation-mediated PCR is commonly applied in DNA sequencing, genome walking, and DNA footprinting.

19. Long-Range PCR

Long-Range PCR is a technique designed to amplify longer DNA fragments that are difficult or impossible to amplify using standard PCR methods or reagents.
It utilizes specially modified, high-efficiency DNA polymerases with enhanced binding and processivity, enabling accurate amplification of long DNA sequences.
This method allows the rapid and efficient amplification of extended DNA targets while optimizing time and resource use.

20. Methylation-specific PCR (MSP)

Methylation-specific PCR (MSP) is a technique used to detect and analyze DNA methylation patterns, particularly in CpG islands.
The process involves treating DNA with bisulfite, which converts unmethylated cytosines to uracil, while leaving methylated cytosines unchanged.
PCR is then performed using two sets of primers: one specific for methylated DNA and another for unmethylated DNA, allowing selective amplification of each type.
Detection of methylation patterns is critical because excessive methylation of CpG dinucleotides in gene promoters can repress gene expression.

21. Miniprimer PCR

Miniprimer PCR is a PCR method that employs an engineered polymerase along with very short primers, called “miniprimers,” typically 10 nucleotides long.
This technique can reveal novel 16S rRNA gene sequences that might not be detected using standard primers.
It utilizes a thermostable DNA polymerase capable of extending from these short primers (9–10 nucleotides).
Miniprimer PCR enables amplification from smaller primer-binding regions and is particularly useful for targeting highly conserved DNA sequences, such as the 16S rRNA gene in prokaryotes or the 18S rRNA gene in eukaryotes.

22. Multiplex PCR

Multiplex PCR is a molecular biology technique that enables the amplification of multiple DNA targets in a single PCR reaction.
The method uses multiple primer pairs along with a temperature-sensitive DNA polymerase in a thermal cycler.
All primer pairs must be carefully optimized so that they share an optimal annealing temperature during the PCR process.
Targeting multiple sequences simultaneously allows the collection of additional information from a single test run, reducing reagent usage and saving time and effort compared to performing separate reactions.
Multiplex PCR is widely applied in genotyping, mutation and polymorphism analysis, microsatellite STR analysis, and detection of pathogens or genetically modified organisms.
In diagnostic laboratories, it is particularly useful for detecting different microorganisms that can cause similar types of diseases.

23. Nanoparticle-Assisted PCR (nanoPCR)

Nanoparticle-Assisted PCR (nanoPCR) incorporates small nanoparticles with specific physical properties that enhance PCR performance.
One proposed mechanism is that gold nanoparticles can adsorb some of the DNA polymerase, regulating the amount of active polymerase in the reaction and thereby improving specificity.
Another mechanism suggests that nanoparticles adsorb primer pairs and reduce the melting temperature difference between perfectly paired and mismatched primers, which increases reaction specificity.
NanoPCR offers high sensitivity, specificity, and selectivity, and has been widely applied in virus detection and gene sequencing.

24. Nested PCR

Nested PCR is a modification of conventional PCR that enhances reaction specificity by using two sets of primers to reduce non-specific binding.
The first set of primers binds outside the target DNA, amplifying a larger fragment, while the second set binds specifically within the target sequence.
In the second amplification round, the inner primers selectively amplify only the target DNA.
Nested PCR is valuable for phylogenetic studies and pathogen detection.
The technique provides higher sensitivity, allowing amplification even from samples containing very low amounts of DNA, which is often not achievable with standard PCR.

25. Overlap extension PCR (OE-PCR)

Overlap Extension PCR (OE-PCR), also known as “Splicing by Overlap Extension” (SOEing), is a technique used to join or modify DNA fragments.
It is commonly applied for cloning large or complex DNA fragments, editing cloned genes, or fusing two gene elements together.
The method allows the construction of long DNA sequences from shorter fragments.
OE-PCR is useful for efficient gene cloning, multiple site-directed insertions, deletions, and replacements of large DNA fragments.
It is particularly valuable for site-directed mutagenesis, creation of chimeric molecules, and cloning of large gene segments by splicing smaller pieces together.

26. Real-Time PCR (Quantitative PCR (qPCR))

Real-Time PCR, also known as Quantitative PCR (qPCR) or quantitative real-time PCR, is a technique that combines DNA amplification with quantification of the target DNA in the reaction.
Unlike conventional PCR, which requires gel electrophoresis for product analysis, qPCR allows real-time monitoring of DNA amplification during the exponential phase.
The method relies on fluorescent dyes or fluorescently labeled oligonucleotides to quantify nucleic acid concentration in the sample.
qPCR is widely applied in genotyping, pathogen quantification, microRNA analysis, cancer detection, microbial load assessment, and detection of genetically modified organisms (GMOs).

27. Repetitive sequence-based PCR

Repetitive Sequence-Based PCR (rep-PCR) is a PCR technique that uses primers targeting noncoding repetitive sequences dispersed throughout the bacterial genome.
These repetitive DNA blocks serve as multiple genetic targets for oligonucleotide primers, allowing the generation of unique DNA fingerprints for individual bacterial strains.
Rep-PCR is primarily applied in molecular strain typing of bacteria and is also useful for epidemiological discrimination of different pathogens.

28. Reverse Transcriptase PCR (RT-PCR)

Reverse Transcriptase PCR (RT-PCR) is a PCR modification in which RNA molecules are first converted into complementary DNA (cDNA), which is then amplified by PCR.
The RNA template is reverse-transcribed into cDNA using reverse transcriptase, and the resulting cDNA serves as the template for exponential amplification.
RT-PCR can be performed either as a one-step reaction in a single tube or as a two-step reaction in separate tubes; the one-step method reduces contamination risk and minimizes variation.
RT-PCR is widely applied in research, gene insertion studies, diagnosis of genetic diseases, and cancer detection.

29. Reverse-Transcriptase Real-Time PCR (RT-qPCR)

Reverse-Transcriptase Real-Time PCR (RT-qPCR) combines reverse transcription PCR (RT-PCR) with quantitative real-time PCR (qPCR).
This technique enables the conversion of RNA into cDNA followed by real-time monitoring and quantification of the amplified DNA during the PCR reaction.

30. RNase H-dependent PCR

RNase H-dependent PCR uses primers that carry a removable amplification block at their 3’ end.
Amplification by the blocked primer occurs only when the RNase H enzyme cleaves the block upon hybridization to the complementary target sequence.
RNase H has minimal activity at low temperatures, providing a natural hot-start effect without modifying the DNA polymerase.
The enzyme’s cleavage efficiency decreases in the presence of mismatches near the RNA residue, reducing non-specific binding and primer-dimer formation.
This mechanism ensures more effective and specific primer hybridization during PCR.

31. Single Specific Primer-PCR (SSP-PCR)

Single Specific Primer PCR (SSP-PCR) enables amplification of double-stranded DNA even when sequence information is available for only one end.
This method allows amplification of genes with only partial sequence knowledge and facilitates unidirectional genome walking from known into previously unknown regions of the chromosome.

32. Solid Phase PCR

Solid-Phase PCR (SP-PCR) is a PCR technique in which one or both primers are immobilized on a solid surface, allowing amplification of target nucleic acids directly on that support.
Spatial separation of primers reduces undesirable interactions, minimizing primer-dimer formation and enabling higher multiplexing capability.
In this method, the 5′-end of primers is attached to the surface rather than allowing primers to freely diffuse in solution.
A freely diffusing DNA target binds to the surface-bound primer and is copied by the polymerase.
The newly synthesized copy remains attached to the surface, while the original DNA molecule returns to the solution after the annealing step.
The free end of the surface-bound copy hybridizes to another surface-attached primer complementary to its sequence, allowing the amplification process to continue.

33. Suicide PCR

Suicide PCR is a PCR method designed to maximize specificity and avoid false positives in sensitive studies.
It requires the use of a primer combination only once, which must never have been used in any prior positive control PCR.
The primers target a genomic region that has not been previously amplified with this or any other primer set, ensuring no carryover contamination from earlier PCR reactions.
This approach prevents false-positive results caused by contaminating DNA in the laboratory.
Suicide PCR is particularly used in paleogenetics, for analyzing preserved genetic material from ancient organisms.

34. Thermal asymmetric interlaced PCR (TAIL-PCR)

Thermal Asymmetric Interlaced PCR (TAIL-PCR) is a technique used to recover DNA fragments adjacent to known sequences.
The method employs three nested primers in consecutive reactions along with an arbitrary degenerate primer with a low melting temperature, allowing thermal control over the relative amplification of specific versus non-specific products.
TAIL-PCR is highly accurate, enabling direct sequencing of unpurified PCR products.
It also facilitates the cloning of full-length functional genes.

35. Touch down PCR

Touchdown PCR is a PCR technique in which the initial annealing temperature is set higher than the primers’ optimal melting temperature (Tm) and is gradually decreased in subsequent cycles until it reaches the target Tm, or “touchdown temperature.”
This approach enhances reaction specificity during the early cycles at higher temperatures and improves amplification efficiency in later cycles as the annealing temperature is lowered.

36. Variable Number of Tandem Repeats (VNTR) PCR

Variable Number of Tandem Repeats (VNTR) PCR is used to amplify DNA fragments that vary between individuals or species, making them valuable markers for forensic identification.
VNTR PCR targets regions that show little variation within a species but differences between species.
The technique can successfully amplify DNA from very small amounts of genomic material.
Among genotyping tools, PCR-based VNTR analysis is a promising method for typing organisms such as Mycobacterium tuberculosis.

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