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.
Porella : features, morphology, anatomy, reproduction etc.
PCR Types
1.
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
12/6/2016 3
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
12/6/2016 5
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
12/6/2016 6
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
12/6/2016 8
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
12/6/2016 9
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 +/+, +/-, -/-
12/6/2016 11
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
12/6/2016 12
13. STEPS
DNA sample
Add Primer of Alu segment
Run PCR
Result on Gel Eletrophoresis
12/6/2016 13
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
12/6/2016 14
15. Steps
DNA sample
Add two primers of different
concentration
Run PCR
Result on Gel Electrophoresis
12/6/2016 15
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
12/6/2016 16
17. Steps
Small quantities of bacterial cells from bacterial colonies are directly added
Add desired Primers for amplification
Run PCR
Results on Gel Electrophoresis
12/6/2016 17
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
12/6/2016 18
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
12/6/2016 19
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
12/6/2016 20
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
12/6/2016 21
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.
12/6/2016 22
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.
12/6/2016 23
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
12/6/2016 24
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
12/6/2016 25
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
12/6/2016 26
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.
12/6/2016 27
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
12/6/2016 28