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.