There are many types of polymerase chain reaction (PCR) that can be classified in different ways. Some of the major types include hot-start PCR, which prevents primer dimer formation; multiplex PCR, which amplifies multiple targets simultaneously; nested PCR, which uses two rounds of PCR for greater specificity; and ligation-mediated PCR, which uses linkers ligated to DNA fragments before amplification. PCR has various applications and techniques that optimize the process for different needs.

1. TYPES OF
POLYMERASE CHAIN
REACTIONS
Apeh Daniel O.
14TH DEC. 2012

2. INTRODUCTION
PCR is an in vitro technique that uses a few
basic everyday molecular biology reagents to
make large numbers of copies of a specific DNA
fragment or a specific region of a DNA strand in
a test-tube.
DNA amplification has several applications
The types are numerous and unclassified
Possible Bases for Classification
Specificity/Error elimination
Where they take place
Application areas
Target
Similarity in methodology
Environmental factors e.t.c

3. REQUIREMENTS FOR PCR
DNA Template
Primers
Taq polymerase
Deoxynucleoside
triphosphates(dNTPs)
Buffer solution
Divalent cations(eg.Mg2+ )
3

4. NEW AUTOMATED PCR OLD PCR
4

5. Types of PCR
1. Hot-Start PCR 15. Ligation-mediated PCR
2. Inverse PCR 16. Methylation-specific PCR (MSP)
3. Multiplex PCR 17. Long PCR
4. Nested PCR 18. Miniprimer PCR
5. Ligation-Mediated PCR 19. Overlap-extension PCR
6. Methylation-Specific PCR (MSP) 20. Quantitative PCR (Q-PCR)
7. Multiplex Ligation-Dependent 21. Reverse Transcription PCR (RT-
Probe Amplification (MLPA) PCR
8. Thermal Asymmetric Interlaced 22. Solid Phase PCR
PCR (Tail- PCR) 23. Touchdown PCR (Step-down PCR)
9. Assembly PCR 24. Universal Fast Walking
10. Asymmetric PCR 25. Variable Number of Tandem
11. Colony PCR Repeats (VNTR) PCR
12. Helicase-dependent amplification 26. InterSequence-Specific PCR (or
13. In Situ PCR (ISH) ISSR-PCR)
14. Intersequence-specific PCR (ISSR)

6. Inverse PCR
 In this method amplification of DNA of unknown sequence is carried out from
known sequence.
 This is especially useful in amplifying and identifying flanking sequences of
various genomic inserts.
Source: Randy et al., 2007

7. Hot start PCR
 Eliminates production of primer dimers caused by
primer annealing at low temperature (55-56 C) before
the start of thermocycling.
 A technique that reduces non-specific amplification
during the initial set up stages of the PCR
 Antibodies or covalently bound inhibitors are used to
inhibit polymerase activity at ambient temperature
 Taq DNA polymerase, for example, Amplitaq Gold which
is activated only if the reaction mixture is heated at
about 94 C

8. Source: Sujatha R (2011) et al 2011

9. Multiplex PCR
Technique for amplification of multiple targets
in a single PCR experiment
Uses multiple pairs of primers to amplify many
sequences simultaneously.
Primers are designed to have similar annealing
temperatures.
Saves time and effort
Applied in Mutation Analysis, Gene Deletion
Analysis

10. Source: Engelstad et al., 2003

11. Nested PCR
 This PCR increases the specificity of DNA
amplification
 Two sets (instead of one pair) of primers are used in
two successive PCRs
 In the first reaction, one pair of primers “outer pair”
is used to generate DNA products
 The product(s) are then used in a second PCR after
the reaction is diluted with a set of second set
“nested or internal” primers whose binding sites are
completely or partially different.
 The specificity of PCR is determined by the specificity
of the PCR primers

12. Source: Fraga MF and Esteller M (2002)

13. Ligation-Mediated PCR
• This method uses small DNA oligonucleotide
'linkers' (or adaptors) that are first ligated to
fragments of the target DNA.
• PCR primers that anneal to the linker
sequences are then used to amplify the target
fragments.
• This method is deployed for DNA
sequencing, genome walking, and DNA
footprinting A related technique is Amplified
fragment length polymorphism, which
generates diagnostic fragments of a genome

14. Source: Engelstad et al., 2003

15. OTHER PCR TYPES WORTHY OF NOTE
• Helicase-dependent amplification: similar to traditional
PCR, but uses a constant temperature rather than cycling
through denaturation and annealing/extension cycles.
DNA helicase, an enzyme that unwinds DNA, is used in
place of thermal denaturation.
• Colony PCR the screening of bacterial (E.Coli) or yeast
clones for correct ligation or plasmid products. Selected
colonies of bacteria or yeast are picked with a sterile
toothpick or pipette tip from a growth (agarose) plate.
This is then inserted into the PCR master mix or pre-
inserted into autoclaved water. PCR is then conducted to
determine if the colony contains the DNA fragment or
plasmid of interest.

16. Conclusion
• Numerous types of PCR exist and the basis on
which they exist vary; ranging from their
application areas, specificity, amplification
target e.t.c. These all combined, gives us an
array of impossibilities in exploitation of genes
in various fields of life.