MB504 Highlighted Handouts Complete PPT by Muhammad Zaman

PCR

 A technique widely used in Molecular Biology and Biotechnology.  Its name is from one of its key component

Polymerase Chain Reaction

PCR

 As PCR progresses, DNA generated is itself used as template for replication.

Polymerase Chain Reaction · PCR - A CHAIN · REACTION

 This sets in motion of a chain reaction in which the DNA template is exponentially amplified.

Polymerase Chain Reaction · HEAT STABLE · DNA

During PCR, a heat stable DNA polymerase, such as Taq polymerase - an enzyme derived from the bacterium Thermus aquaticus .

Polymerase Chain Reaction

 PCR can be performed to amplify DNA.  It can be extensively modified to perform a wide array of genetic manipulations.

Polymerase Chain Reaction · (PPTz Merged by Talha Waraich) · THREE STEPS

 Denaturation  Annealing  Extension

INITIALIZATION STEP

 This step consists of heating the reaction to a temperature of 93 °C - 96 °C, which is held for 110 minutes.

DENATURATION

 First regular cycling event and consists of heating the reaction to 93 °C - 98 °C for 20 45 seconds.  It causes melting of

yielding single strands of DNA.

ANNEALING

temperature is lowered to 40 -65 °C for 2040 seconds.  Annealing of the primers to the single stranded DNA template.

EXTENSION

 The temperature at this step depends on the type of DNA polymerase used.  Taq polymerase has its optimum activity temperature at 70 80 °C.  Commonly 72 °C is used.

EXTENSION

 At this step the DNA

synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's.

FINAL

ELONGATION

 Single step is performed at a temperature of 70 74 °C for 515 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

FINAL HOLD

 This step at 415 °C for an indefinite time may be employed for short-term storage of

INGREDIENTS · OF PCR · MAJOR · INGREDIENTS

 Microfuge tube  Thermal cycler

 Primers  Buffer  MgCl2

MAJOR INGREDIENTS

 Distilled water  Deoxynucleotide triphosphates