POLYMERASE CHAIN REACTION (PCR)

Prepared by

Dr. Naglaa Ibrahim AzabAssistant professor of Medical

Biochemistry

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

• 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.

DNA & RNA EXTRACTION

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,vaginal

discharge,semen hair follicles, bone ,tooth- Viruses present in nucleated cells as WBCs or

cell free in the plasma or serum or body fluids

DDNA

DNAwith histone proteins

m,tRNA

Intracellular organelles&components

Cell membrane

Nuclear membrane

Steps of extraction of DNA

1- Lysis of cell and nuclear membranes2-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.

• 3-precipitation of DNA by alchol and 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.

DNA or RNA extraction using spin column technique

Blood DNA Purification Kit: short protocol

http://www.taq-dna.com/blood-dna-miniprep-_398.html

POLYMERASE CHAIN REACTION(PCR)

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)

Steps of PCR

PCR is amplification of DNA in a tube

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

Put PCR tubes in the PCR machine (Thermal cycler)

Close the thermal cycler

What is the role of the thermal cycler??

Just heats and cools the reaction tubes

After closing the thermal cycler run the program that was saved on the thermal cycler for DNA

amplification in the test tube (PCR)

AT THE END OF PROGRAM YOU HAVE OBTAINED MILLIONS OF

COPIES OF DNA

But How to know that you have amplified the required DNA

SEE YOU NEXT WEEK

THANK YOU