PCR & PCR &

REAL TIME PCRREAL TIME PCR

Submitted By;Submitted By;

Usman KhalidUsman Khalid

Roll No.27Roll No.27

Ucv&ASUcv&AS

PCRPCR & &

REAL TIME REAL TIME PCRPCR

PCRPCR A method for amplifying (copying) small

amount of DNA in nearly any amount required, starting with a small initial quantity.

An in vitro or cell-free method for synthesizing DNA.

It was invented in 1985 by Kary Mullis (received the Nobel Prize for chemistry in 1993).

REAL TIME PCRREAL TIME PCR Is a technique used to monitor the progress of

PCR in real time.A fluorescent reporter molecule is used to

monitor PCR as it progresses. Is of two types, - non-specific detection using binding dyes - specific detection using probes

PCR Machine / ThermocyclerPCR Machine / Thermocycler

PCRPCR

Components of PCRo Template DNAo primerso dNTPs (dATP, dTTP, dCTP & dGTP)o Taq DNA polymeraseo MgCl2

o PCR buffer, pH 8

Taq PolymeraseTaq Polymerase

• Enzymes are proteins that speed up reactions without being consumed themselves. The most important enzyme in a PCR reaction is called Taq polymerase (you know it's an enzyme when you see the -ase suffix at the end of the word). A polymerase is an enzyme that attaches molecules together, and we just so happen to want to attach many nucleotides, the building blocks of DNA, together, so it works out for us.

Taq PolymeraseTaq Polymerase

• Every cell that has DNA (so, pretty much every cell ever) has its own polymerase that takes care of replication of DNA. PCR uses a polymerase from a species of bacteria, Thermus aquaticus, which normally lives in hot springs.

PCRPCR

Three major phases in PCR: o Denaturing (94ºC)o Annealing (55ºC)o Extension (72ºC)

The total time to perform a standard PCR is approximately 4 hours.

Factors influencing PCRFactors influencing PCR

• Quality of template DNA• Concentration of template DNA• Primers• Concentration of MgCl2

• Annealing temperature

Quality of template DNAQuality of template DNA

Should be free of proteases that could degrade the DNA polymerase.

Template DNA with high levels of proteins or salts should be diluted or cleaned up to reduce inhibition of DNA polymerase activity.

Concentration of template DNAConcentration of template DNA

Highly concentrated template DNA may yield nonspecific product or inhibit the reaction.

It is rare that template DNA concentration is too low.

PrimersPrimers Select primers with a random base

distribution and GC content similar to template DNA being amplified.

Avoid sequences with secondary structure, especially at the 3’ end.

Check primers for complementary and avoid primers with 3’ overlaps to reduce primer-dimer artifacts.

Design so the base at the 3’ end of the primer is a G or C to enhance specificity.

Concentration of MgClConcentration of MgCl22

MgCl2 concentration is very important.

Excess Mg2+ promotes production of nonspecific product and primer-dimer artifacts.

Insufficient Mg2+ reduces yield.

Annealing temperatureAnnealing temperature

Annealing temperature depends on length and GC content of primers (55ºC good for primers 20 nucleotides long; 50%).

Higher annealing temperatures may be needed to increase primer specificity.