polymerase chain reaction - pcr
TRANSCRIPT
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PCRPolymerase Chain Reaction
Ahmad A. Al-Qudah
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PCRPolymerase Chain Reaction
- Definitions .- Polymerase Chain Reaction History .- Advantages & disadvantages .- Types of PCR : - Quantitative PCR ( Real Time-PCR ). - Reverse Transcriptase-PCR . - Less Common .
- Polymerase Chain Reaction Principle .- Applications of PCR .- PCR Technologies Bright Future . - Bibliography .
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PCRPolymerase Chain Reaction
Definitions
is a Molecular Biochemical technology, Used to Amplify a single piece of DNA, By a series of Heating and Cooling cycles , generating millions of copies of a particular DNA sequence In Vitro .
- DNA template : contains the DNA region (target) to be amplified .
- DNA primers : complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target .
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- DNA polymerase : Heat-Stabile polymerase , Synthesize the New DNA .
- Nucleotides: the building-blocks of DNA from which the DNA polymerase synthesizes a new DNA strand.
PCRPolymerase Chain Reaction
Definitions
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- PCR was invented in the 1984 as a way to make numerous copies of DNA fragments in the laboratory.
Polymerase Chain Reaction History
- The in vitro version of DNA Replication .
“ Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon ! “
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Polymerase Chain Reaction History
Kary Mullis
Nobel Prize 1993
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Polymerase Chain Reaction Advantages Vs. Disadvantages
* PCR advantages:- Specific amplification .
- Rapid .
- Post-PCR processing of products
- Most specific, sensitive .
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*PCR disadvantages- Setting up and Running requires high technical skills .
- High equipment cost .
- High Test cost .
- High Sterile environment should be provided . (DNA contamination )
Polymerase Chain Reaction Advantages Vs. Disadvantages
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Polymerase Chain Reaction Priciple
Step 1: Denature DNA At 95C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Primers Anneal At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chain At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.
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Polymerase Chain Reaction Principle
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Polymerase Chain Reaction Types of PCR
* Quantitative PCR
- Used to measure the quantity of a target sequence .
- It quantitatively measures starting amounts of DNA, cDNA, or RNA .
- qPCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample .
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Polymerase Chain Reaction Types of PCR
* Quantitative PCR ( Real Time-PCR )
- Quantitative real-time PCR has a very high degree of precision .
- Use fluorescent dyes , or fluorophore-containing DNA probes .
- To measure the amount of amplified product in real time.
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Polymerase Chain Reaction Types of PCR
* Reverse Transcriptase-PCR
- is one of many variants of polymerase chain reaction (PCR) .
- For amplifying DNA from RNA .
- Used to qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA , to detect RNA expression levels .
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Polymerase Chain Reaction Types of PCR
- qPCR is the abbreviation used for Real-Time PCR .
- RT-PCR commonly used as abbreviation for reverse transcription polymerase chain reaction and not real-time PCR .
- Real-time PCR is combined with reverse transcription to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues
RT-PCR Vs. RT-PCR
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Polymerase Chain Reaction Types of PCR
Less Common :- Inverse Polymerase Chain Reaction- Nested polymerase chain reaction- Hot start PCR- Ligation-mediated PCR- Multiplex-PCR- Overlap-extension PCR
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Polymerase Chain Reaction Application of PCR Products
Application of PCR products into one of these Techniques :- Electrophoresis .- Southern Blotting .- Enzyme-Linked Hybridization .- Cross-Linked Hybridization .- FISH .- ASO .- Direct Sequencing .
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Polymerase Chain Reaction Application of PCR
Application of PCR According to the Field of Science :- Medical applications
- Infectious disease applications
- Forensic applications
- Research applications
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Polymerase Chain Reaction Medical applications
- Genetic testing : where a sample of DNA is analyzed for the presence of genetic disease mutations
- Tissue typing : in organ transplantation.
- Oncogenes : Many forms of cancer involve alterations to
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Polymerase Chain Reaction Infectious disease applications
- The Human Immunodeficiency Virus (HIV) : Antibodies to the virus circulating in the bloodstream. don't appear until many weeks after infection, maternal antibodies mask the infection of a newborn . Detection of the Antigen . RT-PCR .
- Tuberculosis : Are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests allowed detection of small numbers of disease organisms (both live or dead) .
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Polymerase Chain Reaction Forensic applications
- Genetic fingerprinting : can uniquely discriminate any person from the entire population of the world. samples of DNA can be isolated from a crime scene , and compared to that from suspects, or from a DNA database of earlier evidence .
- Parental testing : an individual is matched with their close relatives. Less discriminating forms of DNA fingerprinting . DNA compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child .
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Polymerase Chain Reaction Technologies Bright Future
Immunoliposome-PCR
- Several advantages over other immune-PCR methods .
- Ultrasensitive quantitative antigen detection system .
- Reduces false-positive by allows nonspecific DNA inthe assay medium to be degraded .
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Polymerase Chain Reaction Bibliography
- Bartlett, J. M. S.; Stirling, D. (2003). "A Short History of the Polymerase Chain Reaction". PCR Protocols 226. pp. 3–6.
- Saiki, R.; Scharf, S.; Faloona, F.; Mullis, K.; Horn, G.; Erlich, H.; Arnheim, N. (1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science 230(4732): 1350–1354
- Salis AD (2009). "Applications in Clinical Microbiology".Real-Time PCR: Current Technology and Applications. Caister Academic Press
- Pavlov, A. R.; Pavlova, N. V.; Kozyavkin, S. A.; Slesarev, A. I. (2004). "Recent developments in the optimization of thermostable DNA polymerases for efficient applications".Trends in Biotechnology 22 (5): 253–260.
- Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20 (7): 1717–1723.
- http://en.wikipedia.org/wiki/
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