PCR Types

This Presentation contains Eight types pf PCR variants including AFLP, Alu, Asymmetric, Colony, QC, RACE, RAPD and Real Time PCR

1. 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: [email protected]

2. 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 06-Dec-16 3

3. Introduction

4. 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 06-Dec-16 5

5. 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 06-Dec-16 6

6. Components of PCR 06-Dec-16 7

7. 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 06-Dec-16 8

8. Amplified Fragment Length Polymorphism (AFLP) PCR First described by Vos and Zabeau in 1993 P olymorphism in different individuals One Gene having different Alleles of it Amplify the same gene from different individual I dentification 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 06-Dec-16 9

9. 06-Dec-16 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

10. Alu PCR Alu region discoverd by Schmid and Deininger in 1975 Alu sequence on 16 no. chromosome of H uman 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 +/+, +/-, -/- 06-Dec-16 11

11. 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 06-Dec-16 12

12. Steps DNA sample Add Primer of Alu segment Run PCR Result on Gel Eletrophoresis 06-Dec-16 13

13. Asymmetric PCR Direct sequencing and hybridization probing Amplifies just one strand of the target DNA First produce D ouble stranded DNA Then to produce single stranded DNA Unequal primer concentrations A mplification become slow after the one Primer used so Increase cycles 06-Dec-16 14

14. Steps DNA sample Add two primers of different concentration Run PCR Result on Gel Electrophoresis 06-Dec-16 15

15. 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 06-Dec-16 16

16. Steps Small quantities of bacterial cells from bacterial colonies are directly added Add desired Primers for amplification Run PCR Results on Gel Electrophoresis 06-Dec-16 17

17. 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 06-Dec-16 18

18. 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 06-Dec-16 19

19. 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 06-Dec-16 20

20. 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 06-Dec-16 21

21. 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. 06-Dec-16 22

22. 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. 06-Dec-16 23

23. 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 06-Dec-16 24

24. 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 06-Dec-16 25

25. 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 06-Dec-16 26

26. 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. 06-Dec-16 27

27. 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 06-Dec-16 28

28. 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 06-Dec-16 29

29. 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 06-Dec-16 30