TYPES OF PCRAFLP, ALU, ASYMMETRIC,

COLONY, QC, RACE, RAPD AND REAL TIME PCR

Muhammad Usman MughalResearch Scholar

Department of Botany University of the Punjab Lahore

Email: musmanmughal52@yahoo.com

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CONTENTS Introduction

Principle PCR

Components of PCR

Types of PCR

Amplified Fragment Length Polymorphism (AFLP) PCR

ALU PCR

Asymmetric PCR

Colony PCR

Rapid amplification of cDNA ends (RACE) PCR

Quantitative Competitive (QC) PCR

Random Amplified Polymorphic DNA (RAPD) PCR

Real-Time PCR

Conclusion

References

INTRODUCTION

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INFORMATION ABOUT GENERAL PCR

Polymerase Chain Reaction (PCR) was invented by Dr. Kary Mullis in 1983

1993 Dr. Kary Mullis was awarded the Nobel Prize for the discovery of PCR

He shared Nobel Prize in chemistry with Michael Smith

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PRINCIPLE OF PCR

PCR uses the enzyme DNA polymerase to synthesize our desire DNA from template DNA by Adding dNTPs

DNA polymerase is usually Taq Polymerase, extract from bacteria named Thermus aquaticus

Taq polymerase add nucleotides to the 3` end of Primer and complete the region

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COMPONENTS OF PCR

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TYPES OF PCR Amplified Fragment Length Polymorphism (AFLP) PCR

ALU PCR

Asymmetric PCR

Colony PCR

Rapid amplification of cDNA ends (RACE) PCR

Quantitative Competitive (QC) PCR

Random Amplified Polymorphic DNA (RAPD) PCR

Real-Time PCR

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AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) PCR First described by Vos and Zabeau in 1993

Polymorphism in different individuals

One Gene having different Alleles of it

Amplify the same gene from different individual

Identification of genetic changes in strains or closely related species of plants, fungi, animals, and bacteria.

In criminal and paternity tests, Information about populations to generate genetic maps

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Digestion of DNA into Fragments by Restriction Enzymes

Primers ligate on the fragments, these primers area called Adaptors

Amplification of the desired fragments by PCR using two primers

These Primers are complementary of Adaptors

Run PCRAnalysis of the fragments by using gel electrophoresis

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ALU PCR Alu region discoverd by Schmid and Deininger in 1975

Alu sequence on 16 no. chromosome of Human and Primates

Very conserved region

Alu sequences are non-coding, repetitive, about 10,00,000 copies present and it becomes 10%of Human Genome

2 Types of Sequence 731 bp (+) and 416 bp (-)

Genotype +/+, +/-, -/-

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Used for population genetics, Paternity and forensic purpose

Breast cancer, Familial hypercholesterolemia, Hemophilia, Neurofibromatosis Diabetes mellitus type II, Alzheimer's disease, Lung cancer, Gastric cancer

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STEPS DNA sample

Add Primer of Alu segment

Run PCR

Result on Gel Eletrophoresis

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ASYMMETRIC PCR Direct sequencing and hybridization probing

Amplifies just one strand of the target DNA

First produce Double stranded DNA

Then to produce single stranded DNA

Unequal primer concentrations

Amplification become slow after the one Primer used so Increase cycles

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Steps

DNA sample

Add two primers of different concentration

Run PCR

Result on Gel Electrophoresis

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COLONY PCR Colony PCR for determining the presence or absence of insert

DNA in plasmid of Bacteria

Size of the DNA sequence

Biotechnology Products

No need for Extraction and culturing of DNA or plasmid purification steps

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Steps

Small quantities of bacterial cells from bacterial colonies are directly added

Add desired Primers for amplification

Run PCR

Results on Gel Electrophoresis

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RAPID AMPLIFICATION OF CDNA ENDS (RACE) PCR

For obtaining information or Full length sequence of an mRNA

To make map of unique genomic region

Diagnose disease

Steps

cDNA copy of the RNA sequence of interest produced using mRNA

Through reverse transcription

A homopolymeric tail used

PCR amplification of the cDNA copies by PCR

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The copied region is bounded by the known sequence, at either the 5' or 3' end 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR)

Sometime called one-sided PCR or anchored PCR

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QUANTITATIVE COMPETITIVE (QC) PCR Quantitative Competitive PCR is used to measure or quantify the specific

amount of target DNA (or RNA) in a sample

co-amplification of the sequence of interest with diluted synthetic DNA fragment of known concentration which is called competitor

The initial quantity of target molecules in the sample is calculated from the ratio of competitor and amplicons generated during PCR using singlr primer

In this PCR a dilution series of three to five PCR reaction mixtures are made, each with a constant (unknown) amount of added target DNA and a known dilution series of competitor DNA

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The target and competitor DNA compete for the same primers when the concentration of each is equivalent, band intensities will be equivalent

The point of equivalence is determined by visual assessment of band intensities or by digital analysis of the gel image

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RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) PCR William et al discovered RAPD in 1990

Taxonomic identity, kinship relationships, genetic diversity, Genetic Mapping, Population and Evolutionary Genetics,

Unlike traditional PCR, RAPD does not require any specific knowledge of the DNA sequence of the target organism.

It requires only one random primer of 10bp for amplification.

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Steps

Add DNA Sample

Primer is 10bp of random sequence

Run PCR

Study result on Gel Electrophoresis

Because primer is random so it will or will not amplify a segment of DNA

Due to problems in experiment reproducibility, many scientific journals do not accept experiments merely based on RAPDs anymore.

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REAL-TIME PCR Real time PCR is adaptation of the PCR method to quantify the

number of copies during PCR

Quantification of gene expression, Diagnostic uses, Clinical quantification and genotyping

Steps

Add DNA Sample

Add desired Primer and Probe

Probe is short sequence complementary to DNA Like Primer

One Side of Probe is Fluorescent Molecule while on Other end Quencher is Present

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Run PCR

Fluorescent Molecule emitting Fluorescent light with each copy Completing

Probe is between Two Primers

And this Fluorescent intensity detected by the Fluorescent detector in the PCR

Graph developed to determine the copies at any point of PCR

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CONCLUSION

There are many other Molecular Techniques

These technique depends upon application and our desire product

Polymerase chain reaction is major component of all these technique

The name of technique is depend on their uniqueness or purpose

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REFERENCES Cardelli, M. (2011). Alu PCR. PCR Protocols, 221-229

Hadrys, H., Balick, M., & Schierwater, B. (1992). Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Molecular ecology, 1(1), 55-63.

Matz, M. V., Alieva, N. O., Chenchik, A., & Lukyanov, S. (2003). Amplification of cDNA ends using PCR suppression effect and step-out PCR. Generation of cDNA libraries: Methods and Protocols, 41-49.

Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., Ramírez-Solis, R., Webster, T. D., ... & Caskey, C. T. (1989). Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proceedings of the National Academy of Sciences, 86(17), 6686-6690.

Gilliland, G., Perrin, S., Blanchard, K., & Bunn, H. F. (1990). Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proceedings of the National Academy of Science USA, 87, 2725-2729.

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Chien A, Edgar DB, Trela JM (1976). Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127: 1550–1557

Gilliland, G., Perrin, S., Blanchard, K., & Bunn, H. F. (1990). Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proceedings of the National Academy of Science USA, 87, 2725-2729.

Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A. Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

Siebert, P. D., & Larrick, J. W. (1992). Competitive PCR. Nature, 359, 557-558

Wolf, C., & Lüthy, J. (2001). Quantitative competitive (QC) PCR for quantification of porcine DNA. Meat science, 57(2), 161-168

Zon LI, Dorman DM, Orkin SH (1989). The polymerase chain reaction colony miniprep. Biotechniques 7: 696-698.

Competitive PCR Guide - Gene-Quantification.info

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https://en.wikipedia.org/wiki/Alu_element

http://www.geneticorigins.org/pv92/aluframeset.htm

http://xxpresspcr.com/what-are-the-different-kinds-of-pcr/?doing_wp_cron=1480450260.4556720256805419921875

http://www.koko.gov.my/CocoaBioTech/PCRxn3.html

http://www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf

http://bitesizebio.com/19922/the-a-z-of-pcr-variants/

http://xxpresspcr.com/what-are-the-different-kinds-of-pcr/

http://cdn.intechopen.com/pdfs/37264.pdf

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http://pharmaxchange.info/press/2011/07/polymerase-chain-reaction-part-iii-variations-or-types-of-pcr-and-future-prospects-of-pcr/

http://link.springer.com/protocol/10.1007%2F978-1-60761-944-4_15

http://2013.igem.org/wiki/images/6/66/Colony_PCR.pdf

http://www.biotecharticles.com/Biotech-Research-Article/Application-of-RAPD-in-Molecular-Biology-813.html