Types of PCR

1. Amplified fragment length polymorphism (AFLP) PCR

2. Allele-specific PCR
3. Alu PCR
4. Assembly PCR
5. Asymmetric PCR
6. COLD PCR
7. Colony PCR
8. Conventional PCR
9. Digital PCR (dPCR)
10. Fast-cycling PCR
11. High-fidelity PCR
12. High-Resolution Melt (HRM) PCR
13. Hot-start PCR
14. In situ PCR
15. Intersequence-specific (ISSR) PCR
16. Inverse PCR
17. LATE (linear after the exponential) PCR
18. Ligation-mediated PCR
19. Long-range PCR
20. Methylation-specific PCR (MSP)
21. Miniprimer PCR
22. Multiplex-PCR
23. Nanoparticle-Assisted PCR (nanoPCR)
24. Nested PCR
25. Overlap extension PCR
26. Real-Time PCR (quantitative PCR or qPCR)
27. Repetitive sequence-based PCR
28. Reverse-Transcriptase (RT-PCR)
29. Reverse-Transcriptase Real-Time PCR (RT-qPCR)
30. RNase H-dependent PCR (rhPCR)
31. Single cell PCR
32. Single Specific Primer-PCR (SSP-PCR)
33. Solid phase PCR
34. Suicide PCR
35. Thermal asymmetric interlaced PCR (TAIL-PCR)
36. Touch down (TD) PCR
37. Variable Number of Tandem Repeats (VNTR) PCR

Description follows in the following pages

1. Amplified fragment length polymorphism
(AFLP) PCR
 It is a PCR-based technique that uses selective amplification of a
section of digested DNA fragments to generate unique fingerprints for
genomes of interest.
 This technique can quickly generate large numbers of marker
fragments for any organism, without prior knowledge of the genomic
sequence.
 AFLP PCR uses restriction enzymes to digest genomic DNA and allows
attachment of adaptors to the sticky ends of the fragments.
 A part of the restriction fragments is then selected to be amplified by
using primers that are complementary to the adaptor sequence.
 The amplified sequences are separated and visualized on denaturing
on agarose gel electrophoresis.
 AFLP PCR is employed for a variety of applications, as to assess
genetic diversity within species or among closely related species, to infer
population-level phylogenies and biogeographic patterns, to generate
genetic maps and to determine relatedness among cultivars.
2. Allele-specific PCR
 Allele-specific polymerase chain reaction (AS-PCR) is a technique
based on allele-specific primers, which can be used to analyze single
nucleotide polymorphism.
 The allele-specific PCR is also called the (amplification refractory
mutation system) ARMS-PCR corresponding to the use of two different
primers for two different alleles.
 One is the mutant set of primers which are refractory (resistant) to the
normal PCR, and the other is the normal set of primers, which are
refractory to the mutant PCR reaction.
 The 3’ ends of these primers are modified such that one set of the
primer can amplify the normal allele while others amplify the mutant
allele.
 This mismatch allows the primer to amplify a single allele.
 It is widely applied in the single gene point mutation detection such as
sickle cell anemia and thalassemia.
 It is also used for the direct determination of ABO blood group
genotypes.
3. Alu PCR
 Alu PCR is a rapid and easy DNA fingerprinting technique based on the
simultaneous analysis of many genomic loci surrounded by Alu repetitive
elements.
 Alu elements are short stretches of DNA initially characterized by the
action of the Arthrobacter luteus (Alu) restriction endonuclease.
 Alu elements are one of the most abundant transposable elements and
found throughout the human genome, and they play a role in the
evolution and have been used as genetic markers
 In Alu PCR, two fluorochrome-labelled primers complementary to
those sequences are used to perform the PCR, and the PCR products are
then analysed by
 Alu insertions have been used in several genetically inherited human
diseases and various forms of cancer. Thus, this PCR plays an essential
role in the detection of these diseases and mutations.
4. Assembly PCR
 Assembly PCR is a method for the assembly of
large DNA oligonucleotides from multiple shorter fragments.
 In PCR, the size of oligonuleotides used is 18 base pairs, while in
assembly PCR lengths of up to 50bp are used to ensure correct
hybridization.
 During the PCR cycles, the oligonucleotides bind to complementary
fragments and then are filled in by polymerase enzyme.
 Each cycle of this PCR thus increases the length of various fragments
randomly depending on which oligonucleotides find each other.
 Assembly PCR is used to improve the yield of the desired protein and
can also be used to produce large amounts of RNA for structural or
biochemical studies.
5. Asymmetric PCR
 Asymmetric PCR is a variation of PCR used to preferentially amplify
one strand of the original DNA more than the other.
 Asymmetric PCR differs from regular PCR by the excessive amount of
primers for a chosen strand.
 As the asymmetric PCR progresses, the lower concentration 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 is formed after depletion of the limiting primer.
 It is useful when amplification of only one of the two complementary
strands is needed, such as in sequencing and hybridization probing.
6. COLD PCR
 Co-amplification at lower denaturation temperature-based polymerase
chain reaction (COLD-PCR) is a novel form of PCR that selectively
amplifies low-abundance DNA variants from mixtures of wild-type and
 mutant-containing (or variant-containing) sequences, irrespective of the
mutation type or position on the amplicon.
  This method is based on the modification of the critical temperature at
which mutation-containing DNA is preferentially melted over wild type.
 There is an intermediate annealing process after denaturation which
allows hybridization of wild-type and mutant allele. This mismatch
slightly alters the melting temperature of the ds DNA.
 These heteroduplexes will melt and will be used as a template. As s
result, a more significant proportion of minor variant DNA will be
amplified and be available for subsequent rounds of PCR.
 PCR plays a vital role in the detection of mutations in oncology
specimens, especially in heterogeneous tumours as well as bodily fluids.
 This PCR also assists in the assessment of residual disease after
surgery or chemotherapy and disease staging and molecular profiling for
prognosis or tailoring therapy to individual patients.
7. Colony PCR
 Colony PCR is a method in which, where identification of DNA of
interest inserted into the plasmid is obtained by designing the inserted
DNA specific primers.
 The bacterial colony containing the plasmid can directly be amplified
using two sets of primers.
 The first set is of the insert specific primers which amplify the insertion
sequence, and the other is of vector-specific flanking primers, which
amplifies the plasmid DNA other than the inserted DNA.
 A bacterial colony is taken and added directly into the master mix
containing all other PCR reagents.
 The main application of colony PCR is in the identification of correct
ligation and insertion of inserted DNA into bacteria as well as yeast
plasmid.
8. Conventional PCR
 The polymerase chain reaction(PCR) is a test tube system for DNA
replication which allows a “target” DNA sequence to be selectively
amplified several million folds in just a few hours.
 PCR enables the synthesis of specific DNA fragments using a DNA-
polymerase enzyme, which takes part in the replication of the cellular
genetic material.
 This enzyme synthesizes a complementary sequence of DNA, as a
small fragment (primer) is connected to one of the DNA strands in the
specific site chosen to start the synthesis.
 Primers limit the sequence to be replicated, and the result is the
amplification of a particular DNA sequence with billions of copies.
 Conventional PCR is applied in selective DNA isolation, amplification
and quantification of DNA, medical and diagnostic approaches, infectious
disease diagnosis, forensic studies and research areas.
9. Digital PCR (dPCR)
 Digital PCR (dPCR) is a quantitative PCR technology that provides a
sensitive and efficient way for the measurement of the amount of DNA
or RNA present in a sample.
 For dPCR, the initial sample mix is divided into a large number of
individual wells prior to the amplification step, resulting in either target
sequence being present in each well or not.
 Based on the presence or absence of fluorescence in the amplified
reaction wells calculation of the absolute number of targets present in
the original sample is done.
 Wells with a fluorescent signal are considered positives and scored as
“1” while wells with no such signal are negatives and scored as “0”.
 The concentration of the target sequence present in the initial sample
is then determined through Poisson statistical analysis.
 dPCR is used to determine the total numbers of DNA and RNA viruses,
bacteria, and parasites in a variety of clinical specimens, mainly when a
well-calibrated standard is not available.
10. Fast cycling PCR
 Fast cycling PCR is a PCR-based technology that allows amplification of
specific PCR products with significantly reduced cycling time.
 The principle in this process is the same as conventional PCR, the only
difference being the time of amplification.
 The buffer used in this PCR increases the affinity of Taq DNA
polymerases for short single-stranded DNA fragments, reducing the time
required for successful primer annealing to just 5 seconds.
 Fast cycling PCR is essential for processes requiring quick cycles and
also helps in the rapid diagnosis of diseases and mutations.
11. High Fidelity PCR
 High-fidelity PCR is a modifies PCR method that utilizes a DNA
polymerase with a low error rate and results in a high degree of
accuracy in the replication of the DNA of interest.
 Such enzymes have a significant binding affinity for the correct
nucleoside triphosphate during amplification.
 In the case of an incorrect binding in the polymerase active site,
incorporation is slowed due to the architecture of the active site
complex.
 Highfidelity amplification is essential for experiments whose outcome
depends upon the correct DNA sequence like cloning, SNP analysis, NGS
applications.
12. High-Resolution Melt (HRM) PCR
 It is a hugely powerful technique for the detection of mutations,
polymorphisms and epigenetic differences in double stranded DNA
samples.
 It is massively cost-effective vs. other genotyping technologies such as
sequencing and Taqman SNP typing. This makes it ideal for large scale
genotyping projects.
 It is fast and powerful thus able to accurately genotype huge numbers
of samples in rapid time.
 It is simple. With a good quality HRM assay powerful genotyping can
be performed by non-geneticists in any laboratory with access to an
HRM capable real-time PCR machine.
13. Hot start PCR
 Hot start PCRis a novel form of conventional polymerase chain reaction
(PCR) that reduces the occurrence of undesired products and formation
of primer-dimers due to non-specific DNA amplification at room
temperatures.
 The basic principle of hot-start PCR is the separation of one or more
reagents from the reaction mix until the mixture reaches the
denaturation temperature upon heating.
 Hot start PCR significantly reduces non-specific binding, the formation
of primer-dimers, and often increases product yields. It also requires
less effort and reduces the risk of contamination.
14. In-situ PCR
 In-Situ Polymerase Chain Reaction(In-situ PCR) is an effective method
that detects minute quantities of rare nucleic acid sequences in frozen or
paraffin-embedded cells or tissue sections for the compartmentalization
of those sequences within the cells.
 This method involves tissue fixing that preserves the cell morphology,
which is then followed by the treatment with proteolytic enzymes to
provide an entry for the PCR reagents to act on the target DNA.
 The target sequences are amplified by the reagents and then detected
by standard immunocytochemical protocols.
 In-situ PCR is applicable for the diagnosis of infectious diseases,
quantification of DNA, detection of even small amount of DNA and is
widely used in the study of organogenesis and embryogenesis.
15. Intersequence specific (ISS) PCR
 InterSequence-Specific PCR (or ISSR-PCR) is a method for DNA
fingerprinting that uses primers selected from specific segments
repeated throughout a genome to produce a unique fingerprint.
 The technique uses microsatellites, usually 16–25 bp long, as primers
in a single primer PCR reaction targeting multiple genomic loci to amplify
mainly the inter- SSR sequences of different sizes.
 ISSR PCR can be used in genomic fingerprinting, genetic diversity and
phylogenetic analysis, genome mapping and gene tagging.
16. Inverse PCR
 Inverse polymerase chain reaction (Inverse PCR) is one of the many
variants of the polymerase chain reaction that is used to amplify DNA
when only one sequence is known.
 Conventional PCR requires primers complementary to both terminals
of the target DNA, but Inverse PCR allows amplification to be carried out
even if only one sequence is available from which primers may be
designed.
 The inverse PCR involves a series of restriction digestion followed by
ligation, which results in a looped fragment that can then be primed for
PCR through a single section of known sequence.
 Then, like other polymerase chain reaction processes, the DNA is
amplified by the temperature-sensitive DNA polymerase.
 Inverse PCR is especially useful for the determination of insert
locations of various transposons and retroviruses in the host DNA.
17. LATE (Linear-After-The-Exponential)
PCR
 LATE (Linear-After-The-Exponential) PCR is a modification of
Asymmetric PCR which uses a limiting primer with a higher melting
temperature than the excess primer which maintains reaction efficiency
as the limiting primer concentration decreases mid-reaction.
 LATE-PCR begins with an exponential phase in which amplification
efficiency is similar to that in conventional PCR. Once the limiting primer
is depleted, the reaction abruptly switches to linear amplification, and
the single-stranded product is continued for many additional thermal
cycles.
18. Ligation mediated PCR
 Ligation-mediated PCR is a modified form of conventional PCR that is
possible with the knowledge of only one end initially and then adding the
second end by ligation of a unique DNA linker.
 Ligation-mediated PCR utilizes small DNA fragments called ‘linkers’ (or
adaptors) that are initially ligated to fragments of the target DNA.
 PCR primers designed to bind to the linker sequences are then used to
amplify the target fragments.
 This method is deployed for DNA sequencing, genome walking, and
DNA footprinting.
19. Long-Range PCR
 Long-Range PCR is a method for the amplification of longer DNA
lengths that cannot typically be amplified using routine PCR methods or
reagents.
 Long-range PCR can be achieved by using modified high-efficiency
polymerases with enhanced DNA binding, resulting in highly processive
and accurate amplification of long fragments.
 This method allows the amplification of more extended targets within a
shorter period and with efficient use of resources.
20. Methylation-specific PCR (MSP)
 Methylation-specific PCR (MSP) is a method for the detection and
analysis of DNA methylation patterns in CpG islands.
 For performing MSP, DNA is modified by, and PCR performed with two
primer pairs, which are detectable methylated and unmethylated DNA,
respectively.
 The DNA undergoes treatment with bisulfite for the conversion of
cytosine to uracil, and then the methylated sequences are selectively
amplified with primers specific for 
 Detection of methylated patterns is essential as excessive methylation
of CpG dinucleotides in promoter represses the gene expression.
21. Miniprimer PCR
 A new PCR method using an engineered polymerase and 10-nucleotide
“miniprimers” is termed Miniprimer PCR.
 This method is found to reveal novel 16S rRNA gene sequences that
would not have been detected with standard primers.
 Miniprimer PCR  uses a thermostable polymerase enzyme that can
extend from short primers (9 or 10 nucleotides).
 This method allows PCR targeting to smaller primer binding regions,
and is used to amplify highly conserved DNA sequences, such as the 16S
(or eukaryotic 18S) rRNA gene.
22. Multiplex PCR
 Multiplex PCR is a common molecular biology technique used for the
amplification of multiple targets in a single PCR test run.
 In Multiplex PCR, multiple primers and a temperature-mediated DNA
polymerase are used for the amplification of DNA in a thermal cycler.
 All the primers pairs designed for Multiplex PCR have to be optimized
so that the same annealing temperature is optimal for all the pairs
during PCR.
 When multiple sequences are targeted at once, additional information
can be generated from a single test run which otherwise would require a
larger amount of the reagents and extensive time and effort to perform.
 This technology has been applied in many areas such as genotyping,
mutation and polymorphism analysis, microsatellite STR analysis,
detection of pathogens or genetically modified organisms, etc.
 In diagnostic laboratories, multiplex PCR is useful to detect different
microorganisms that cause the same types of diseases.
23. Nanoparticle-Assisted PCR (nanoPCR)
 A nanoparticle associated PCR includes small molecular substances
comprising of particular physical properties that enhance the reaction.
 One of the theories involving the gold nanoparticles states that these
particles adsorb some of the polymerase and manages the amount of
polymerase remaining in the system, which might be necessary in
enhancing the specificity of the reaction.
 Another theory explains that they adsorb primer pairs and lower the
melting temperature at duplex formation between perfectly paired and
mispaired primers, which leads to an increase in the specificity of the
reaction.
 Nanoparticle associated PCR has advantages of high sensitivity, high
specificity and high selectivity, and has been widely used in virus
detection and gene sequencing.
24. Nested PCR
 Nested PCR is a useful modification of PCR technology where the
specificity of the reaction is enhanced by preventing the non-specific
binding with the help of the two sets of primer.
 The first set of primer binds outside of our target DNA and amplifies
larger fragment while another set of primer binds specifically at the
target site.
 In the second round of amplification, second set of primer amplifies
only the target DNA.
 Nested PCR is a helpful method for the phylogenetic studies and
detection of different pathogens.
 The technique has higher sensitivity; hence even if the sample
contains lower DNA, it can be amplified which is not feasible in the
conventional PCR technique.
25. Overlap extension PCR (OE-PCR)
 This method is also called “Splicing by Overlap Extension” or SOEing.
 Overlap extension PCR is a valuable technique that is commonly used
for cloning large complex fragments, making edits to cloned genes or
fusing two gene elements together.
 It creates long DNA fragments from shorter ones.
 It is used for efficient gene cloning and multiple site-directed large
fragments insertion, deletion and replacement.
 It is proven useful for site-directed mutagenesis, the creation of
chimeric molecules or even the cloning of large gene segments by
splicing together smaller pieces.
26. Real-Time PCR (Quantitative PCR
(qPCR))
 Quantitative PCR (qPCR), also called real-time PCR or
quantitative real-time PCR, is a PCR-based technique that couples
amplification of a target DNA sequence with quantification of the
concentration of that DNA species in the reaction.
 Conventional PCR is a time-consuming process where the PCR
products are analysed through gel electrophoresis. qPCR facilitates the
analysis by providing real time detection of products during the
exponential phase.
 The principle of real-time PCR depends on the use of fluorescent dye.
 The concentration of the nucleic acid present into the sample is
quantified using the fluorescent dye or using the fluorescent labelled
oligonucleotides.
 q-PCR is applied in genotyping and quantification of pathogens,
microRNA analysis, cancer detection, microbial load testing and GMOs
detection.
27. Repetitive sequence-based PCR
 Repetitive sequence-based PCR (rep-PCR) is a modified PCR
technology that uses primers that target noncoding repetitive sequences
interspersed throughout the bacterial genome.
 Such blocks of noncoding, repetitive sequences can serve as multiple
genetic targets for oligonucleotide probes, enabling the generation of
unique DNA profiles or fingerprints for individual bacterial strains.
 The main application of rep-PCR is in the molecular strain typing of
different bacteria. It is also used for epidemiologic discrimination of
various pathogens.
28. Reverse Transcriptase PCR (RT-PCR)
 Reverse transcription PCR (RT-PCR) is a modification of conventional
PCR, whereby RNA molecules are first converted into complementary
DNA (cDNA) molecules that can then be amplified by PCR.
 In RT-PCR, the RNA template is first converted into a complementary
DNA (cDNA) using reverse transcriptase. The cDNA then acts as a
template for exponential amplification using PCR.
 RT-PCR can be conducted either in a single tube or as two steps in
different tubes. The one-step method is more effective with fewer
chances of contamination and incorporation of variations.
 RT-PCR is used in research methods, gene insertion, genetic disease
diagnosis and cancer detection.
29. Reverse-Transcriptase Real-Time PCR
(RT-qPCR)
 RT-PCR is commonly associated with q-PCR forming Reverse
Transcriptase Real-Time PCR (RT-qPCR).
 This allows quantification of DNA in real-time after the amplification.
30. RNase H-dependent PCR
 In the RNase H-dependent PCR, the primers contain a removable
amplification block on their 3’ end.
 The blocked primer can only perform amplification depending on the
cleavage activity of an RNase Henzyme during hybridization to the
complementary target sequence. 
 RNase H enzyme has very little enzymatic activity at a low
temperature, enables a hot start without any modification to the DNA
polymerase.
 Similarly, the cleavage efficiency of the enzyme is reduced in the
presence of mismatches near the RNA residue.
 Thus, under the activity of the RNase H enzyme, the non-specific
binding and primer dimer formation is reduced, enabling effective
hybridization.
31. Single Specific Primer PCR
 The single specific primer-PCR (SSP-PCR) is a PCR-based technology
that permits amplification of genes of which, only a piece of partial
sequence information is available.
 It allows unidirectional genome walking from known into unknown
regions of the chromosome.
32. Single Specific Primer-PCR (SSP-PCR)
 This allows the amplification of double-stranded DNA even when the
sequence information is available at one end only.
 This method, the single specific primer-PCR (SSP-PCR), permits
amplification of genes for which only a partial sequence information is
available, and allows unidirectional genome walking from known into
unknown regions of the chromosome.
33. Solid Phase PCR
 Solid-phase PCR (SP-PCR) is a unique PCR technique that allows
amplification of target nucleic acids on a solid support where one or both
primers are immobilized on the surface.
 The spatial separation of the primers minimizes significantly
undesirable primer interactions, thereby preventing the formation of
primer-dimers and allowing higher multiplexing amplification.
 The central idea of this novel method is to attach the 5′-end of the
primers to a surface instead of letting the primers freely diffuse in a bulk
solution.
 A freely diffusing DNA target can be captured on the surface and then
copied by the polymerase.
 The copy stays attached to the surface, whereas the initial DNA
molecule returns to the solution after the annealing step.
 The free end of the attached copy hybridizes to the primer (attached
to the surface) complementary to its sequence, and the amplification
process can start.
34. Suicide PCR
 Suicide PCR is commonly used studies where avoiding false positives
and ensuring the specificity of the amplified fragment is the highest
priority.
 The method requires the use of any primer combination only once in a
PCR, which should not have been used in any positive control PCR
reaction.
 These primers should always target a genomic region which has never
been amplified before using this particular primer or any other set of
primers.
 This arrangement ensures that no contaminating DNA from previous
PCR reactions is present in the lab, which could otherwise generate false
positives.
 Suicide PCR is used in paleogenetics studies which involve an
examination of preserved genetic material from the remains of ancient
organisms.
35. Thermal asymmetric interlaced PCR
(TAIL-PCR)
 TAIL PCR is a powerful tool for the recovery of DNA fragments
adjacent to known sequences.
 TAIL –PCR utilizes three nested primers in consecutive reactions
together with an arbitrary degenerate primer having a low melting
 temperature so that relative amplification frequencies of specific and
non-specific products can be thermally controlled.
 This method is highly accurate such that the unpurified TAIL-PCR
products can be directly sequenced.
 It also allows the cloning of full-length functional genes.
36. Touch down PCR
 Touch Down PCR is a modification of PCR in which the initial annealing
temperature is higher than the optimal Tm of the primers and is
gradually reduced over subsequent cycles until the Tm temperature or
“touchdown temperature” is reached.
 Touchdown PCR increases the specificity of the reaction at higher
temperatures and increases the efficiency towards the end by lowering
the annealing temperature.
37. Variable Number of Tandem Repeats
(VNTR) PCR
 They are important markers for the individualization in forensic
science.
 In VNTR PCR, fragments are amplified that showed little variation
within a species, but did show differences between species.
 It can successfully amplify from a very small amount of genomic
deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR).
 Among the genotyping tools, the PCR-based variable-number tandem
repeat (VNTR) analysis represented a promising method for typing M.
tuberculosis.