PCRPolymerase Chain Reaction

Ahmad A. Al-Qudah

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 .

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 .

- 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

- 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 ! “

Polymerase Chain Reaction History

Kary Mullis

Nobel Prize 1993

Polymerase Chain Reaction Advantages Vs. Disadvantages

* PCR advantages:- Specific amplification .

- Rapid .

- Post-PCR processing of products

- Most specific, sensitive .

*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

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.

Polymerase Chain Reaction Principle

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 .

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.

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 .

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

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

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 .

Polymerase Chain Reaction Application of PCR

Application of PCR According to the Field of Science :- Medical applications

- Infectious disease applications

- Forensic applications

- Research applications

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

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) .

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 .

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 .

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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