PCR (polymerase chain reaction) is a technique used to amplify DNA sequences. There are many types of PCR that have different applications. Some types are used to detect genetic mutations (allele-specific PCR), analyze genetic diversity (AFLP PCR), detect DNA in tissues (in situ PCR), amplify long DNA fragments (long-range PCR), or detect rare DNA variants (COLD PCR). Digital PCR allows absolute quantification of DNA by dividing the sample into many individual reactions, while real-time PCR allows quantification during the amplification process. The various PCR techniques have a wide range of uses including disease diagnosis, forensics, and research.

1. Types of PCR (Polymerase Chain Reaction) – Definition And Uses
List of 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)

2. 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
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.

3. • 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.

4. 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.

5. 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.

6. • 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.

7. • 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.

8. • 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.
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.

9. • 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.

10. • 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.

11. • 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.

12. • 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.

13. • 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.

14. • 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.
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