TYPES OF POLYMERASE CHAIN REACTIONS

Apeh Daniel O.

14TH DEC. 2012

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 ClassificationSpecificity/Error eliminationWhere they take placeApplication areasTarget Similarity in methodologyEnvironmental factors e.t.c

INTRODUCTION

DNA TemplatePrimersTaq polymeraseDeoxynucleoside triphosphates(dNTPs)Buffer solutionDivalent cations(eg.Mg2+ )

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REQUIREMENTS FOR PCR

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NEW AUTOMATED PCR OLD PCR

1. Hot-Start PCR2. Inverse PCR3. Multiplex PCR 4. Nested PCR5. Ligation-Mediated PCR6. Methylation-Specific PCR (MSP)7. Multiplex Ligation-Dependent

Probe Amplification (MLPA)8. Thermal Asymmetric Interlaced

PCR (Tail- PCR)9. Assembly PCR10. Asymmetric PCR11. Colony PCR 12. Helicase-dependent amplification13. In Situ PCR (ISH) 14. Intersequence-specific PCR (ISSR)

15. Ligation-mediated PCR 16. Methylation-specific PCR (MSP) 17. Long PCR 18. Miniprimer PCR19. Overlap-extension PCR20. Quantitative PCR (Q-PCR)21. Reverse Transcription PCR (RT-

PCR22. Solid Phase PCR23. Touchdown PCR (Step-down PCR)24. Universal Fast Walking25. Variable Number of Tandem

Repeats (VNTR) PCR 26. InterSequence-Specific PCR (or

ISSR-PCR)

Types of 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.

Inverse PCR

Source: Randy et al., 2007

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

Hot start PCR

Source: Sujatha R (2011) et al 2011

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 effortApplied in Mutation Analysis, Gene Deletion

Analysis

Multiplex PCR

Source: Engelstad et al., 2003

This PCR increases the specificity of DNA amplificationTwo 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 productsThe 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

Nested PCR

Source: Fraga MF and Esteller M (2002)

• 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

Ligation-Mediated PCR

Source: Engelstad et al., 2003

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.

• 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.

Conclusion

Thank you and God bless