POLYMERASE CHAIN REACTION (PCR) Prepared by Dr. Naglaa Ibrahim Azab Assistant professor of Medical Biochemistry
Jan 12, 2016
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
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