This document discusses 8 types of PCR: AFLP, ALU, asymmetric, colony, RACE, QC, RAPD, and real-time PCR. It provides details on the principles, components, and steps of each type. General PCR involves using DNA polymerase to synthesize DNA from a template. The types vary in their application, such as identifying genetic polymorphisms, determining gene expression, and obtaining full-length mRNA sequences.

2. TYPES OF PCR
AFLP, ALU, ASYMMETRIC, COLONY, QC,
RACE, RAPD AND REAL TIME PCR
Muhammad Usman Mughal
Research Scholar
Department of Botany University of the Punjab Lahore
Email: musmanmughal52@yahoo.com

3. 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
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4. INTRODUCTION

5. 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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6. 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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7. COMPONENTS OF PCR
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8. 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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9. 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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10. 12/6/2016 10
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 PCR
Analysis of the
fragments by using
gel electrophoresis

11. 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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12.  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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13. STEPS
 DNA sample
 Add Primer of Alu segment
 Run PCR
 Result on Gel Eletrophoresis
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14. 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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15.  Steps
 DNA sample
 Add two primers of different
concentration
 Run PCR
 Result on Gel Electrophoresis
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16. 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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17.  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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18. 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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19.  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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20. 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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21.  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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22. 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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23.  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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24. 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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25.  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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26. 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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27. 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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28.  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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29.  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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30.  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
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