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POLYMERASE CHAIN REACTION (PCR)

POLYMERASE CHAIN REACTION (PCR). Prepared by Dr. Naglaa Ibrahim Azab Assistant professor of Medical Biochemistry. What is PCR?. PCR is DNA Amplification in vitro

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POLYMERASE CHAIN REACTION (PCR)

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  1. POLYMERASE CHAIN REACTION (PCR) Prepared by Dr. Naglaa Ibrahim Azab Assistant professor of Medical Biochemistry

  2. What is PCR? PCR is DNA Amplification in vitro It has the same idea of DNA replication but in a test tube ( 2 DNA strands seperation, primers anealing and elongation by the DNA polymerase)

  3. FOR AMPLIFICATION OF • DNA: We must extract the pure DNA that is required to be amplified, from the cells or body fluids. • RNA : We must extract the pure RNA that is required to be amplified, from the cells or body fluids. This is followed by conversion of RNA to DNA by reverse transcriptase enzyme as the DNA polymerase can not elongate the RNA, but only DNA.

  4. DNA & RNA EXTRACTION

  5. What are the sources of DNA and RNA?? • Nucleated cells or cell free DNA as - blood cells - buccal cells -solid tissues - forensic samples as body fluids,vaginaldischarge,semen hair follicles, bone ,tooth - Viruses present in nucleated cells as WBCs or cell free in the plasma or serum or body fluids

  6. Cell membrane DDNA Intracellular organelles &components DNAwithhistone proteins m,tRNA Nuclear membrane

  7. Steps of extraction of DNA 1- Lysis of cell and nuclear membranes 2-Removal of cellular and histone proteins by: -organic solvents as phenol or phenol- chloroform mixture( alchol precipitation) -Salt precipitation of proteins using saturated salt solutions - Digestion by enzymes as protease or proteinase k.

  8. 3-precipitation of DNA by alcholand then the excess alchol (supernatent) removed and DNA is rehydrated OR allow the solution to pass through silica which binds DNA . Then wash the DNA from any debris or other material by wash buffer Then elute DNA from silica by elution buffer or nuclease free water.

  9. DNA or RNA extraction using spin column technique

  10. Blood DNA Purification Kit: short protocol

  11. POLYMERASE CHAIN REACTION(PCR)

  12. What is PCR? As already said PCR is DNA Amplification in vitro It has the same idea of DNA replication but in a test tube ( 2 DNA strands seperation, primers anealing and elongation by the DNA polymerase)

  13. Steps of PCR

  14. PCR is amplification of DNA in a tube

  15. What to put in the PCR tube??What is the role of every component?? • Template DNA • Pair of primers • Thermostable DNA polymerase • dNTPs • Buffer • Cations required for the activity of the enzyme usually Mg ions

  16. Put PCR tubes in the PCR machine (Thermal cycler)

  17. Close the thermal cycler

  18. What is the role of the thermal cycler?? Just heats and cools the reaction tubes

  19. After closing the thermal cycler run the program that was saved on the thermal cycler for DNA amplification in the test tube (PCR)

  20. AT THE END OF PROGRAM YOU HAVE OBTAINED MILLIONS OF COPIES OF DNA

  21. But How to know that you have amplified the required DNA SEE YOU NEXT WEEK Thank you

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