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: [email protected]
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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