PCR
Polymerase Chain Reaction
Ahmad A. Al-Qudah
PCR
Polymerase Chain Reaction
- Definitions .
- Polymerase Chain Reaction History .
- Advantages & disadvantages .
- Types...
PCR
Polymerase Chain Reaction
Definitions
is a Molecular Biochemical technology, Used to Amplify a single piece
of DNA, By...
- DNA polymerase : Heat-Stabile polymerase , Synthesize the New DNA .
- Nucleotides: the building-blocks of DNA from which...
- PCR was invented in the 1984 as a way to make numerous
copies of DNA fragments in the laboratory.
Polymerase Chain React...
Polymerase Chain Reaction
History
Kary Mullis
Nobel Prize 1993
Polymerase Chain Reaction
Advantages Vs. Disadvantages
* PCR advantages:
- Specific amplification .
- Rapid .
- Post-PCR p...
*PCR disadvantages
- Setting up and Running requires high technical skills .
- High equipment cost .
- High Test cost .
- ...
Polymerase Chain Reaction
Priciple
Step 1: Denature DNA
At 95C, the DNA is denatured (i.e. the two
strands are separated)...
Polymerase Chain Reaction
Principle
Polymerase Chain Reaction
Types of PCR
* Quantitative PCR
- Used to measure the quantity of a target sequence .
- It quant...
Polymerase Chain Reaction
Types of PCR
* Quantitative PCR ( Real Time-PCR )
- Quantitative real-time PCR has a very high d...
Polymerase Chain Reaction
Types of PCR
* Reverse Transcriptase-PCR
- is one of many variants of polymerase chain reaction
...
Polymerase Chain Reaction
Types of PCR
- qPCR is the abbreviation used for Real-Time PCR .
- RT-PCR commonly used as abbre...
Polymerase Chain Reaction
Types of PCR
Less Common :
- Inverse Polymerase Chain Reaction
- Nested polymerase chain reactio...
Polymerase Chain Reaction
Application of PCR Products
Application of PCR products into one of these
Techniques :
- Electro...
Polymerase Chain Reaction
Application of PCR
Application of PCR According to the Field of
Science :
- Medical applications...
Polymerase Chain Reaction
Medical applications
- Genetic testing :
where a sample of DNA is analyzed for the
presence of g...
Polymerase Chain Reaction
Infectious disease applications
- The Human Immunodeficiency Virus (HIV) :
Antibodies to the vir...
Polymerase Chain Reaction
Forensic applications
- Genetic fingerprinting :
can uniquely discriminate any person from the
e...
Polymerase Chain Reaction
Technologies Bright Future
Immunoliposome-PCR
- Several advantages over other immune-PCR
methods...
Polymerase Chain Reaction
Bibliography
- Bartlett, J. M. S.; Stirling, D. (2003). "A Short History of the Polymerase Chain...
Polymerase Chain Reaction - PCR
Polymerase Chain Reaction - PCR
Polymerase Chain Reaction - PCR
Polymerase Chain Reaction - PCR
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Polymerase Chain Reaction - PCR

Polymerase Chain Reaction - PCR
Published on: Mar 4, 2016
Published in: Health & Medicine      
Source: www.slideshare.net


Transcripts - Polymerase Chain Reaction - PCR

  • 1. PCR Polymerase Chain Reaction Ahmad A. Al-Qudah
  • 2. PCR Polymerase 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 .
  • 3. PCR Polymerase 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 .
  • 4. - 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. PCR Polymerase Chain Reaction Definitions
  • 5. - 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 ! “
  • 6. Polymerase Chain Reaction History Kary Mullis Nobel Prize 1993
  • 7. Polymerase Chain Reaction Advantages Vs. Disadvantages * PCR advantages: - Specific amplification . - Rapid . - Post-PCR processing of products - Most specific, sensitive .
  • 8. *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
  • 9. Polymerase Chain Reaction Priciple Step 1: Denature DNA At 95C, the DNA is denatured (i.e. the two strands are separated) Step 2: Primers Anneal At 40C- 65C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA Step 3: DNA polymerase Extends the DNA chain At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers.
  • 10. Polymerase Chain Reaction Principle
  • 11. 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 .
  • 12. 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.
  • 13. 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 .
  • 14. 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
  • 15. 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
  • 16. 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 .
  • 17. Polymerase Chain Reaction Application of PCR Application of PCR According to the Field of Science : - Medical applications - Infectious disease applications - Forensic applications - Research applications
  • 18. 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
  • 19. 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) .
  • 20. 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 .
  • 21. 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 in the assay medium to be degraded .
  • 22. 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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