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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

POLYMERASE CHAIN REACTION (PCR)

Prepared by

Dr. Naglaa Ibrahim Azab

Assistant professor of Medical Biochemistry

what is pcr
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)

for amplification of
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.
what are the sources of dna and rna
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

slide6

Cell membrane

DDNA

Intracellular organelles

&components

DNAwithhistone proteins

m,tRNA

Nuclear membrane

steps of extraction of dna
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.

slide8

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.

what is pcr1
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)

what to put in the pcr tube what is the role of every component
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
what is the role of the thermal cycler
What is the role of the thermal cycler??

Just heats and cools the reaction tubes

slide23
After closing the thermal cycler run the program that was saved on the thermal cycler for DNA amplification in the test tube (PCR)