Amplification of - PowerPoint Presentation

Slide1

Amplification of a DNA fragment by Polymerase Chain Reaction (PCR)

Ms. Nadia AmaraSlide2

The polymerase chain reaction (PCR) is a powerful and sensitive technique for DNA amplification in vitro .The enormous utility of PCR is based on its ease of use and its ability to amplify DNA.

The PCR

amplification uses an

enzyme known as

Taq

DNA polymerase. This enzyme, originally purified from a bacterium that inhabits hot springs

, is

stable at very high temperatures

.

Also included in

the PCR

reaction mixture are two

synthetic oligonucleotides

(

15-30 nucleotides

) known as "primers" and the region

of DNA

to be amplified is known as the "target" or the "template"

.

These

components, together are incubated in an appropriate buffer that contains Mg2+.Slide3

** The primers are designed to correspond to the start and end of the DNA to be amplified "target".The PCR reaction mixture (which contains the DNA polymerase, buffer, deoxy

nucleotides, primers, and template) is subjected to sequential heating/cooling cycles at three different temperatures .Slide4

The cycling reaction :

1. Denaturation :

In the first step of the PCR reaction, the hydrogen bonds between template complementary DNA strands are separated (denatured) from each other at 94°C, while the

Taq

DNA polymerase remains stable

.

2. Annealing :

In the second step, the sample is cooled to an intermediate temperature (usually 40°- 65°C) to allow hybridization of the two primers to the two strands, one to each of the two strands of the target DNA sequence

.

3. Extension :

In the third step, known as extension (also sometimes called DNA synthesis), the temperature is raised to 72°C and the

Taq

DNA polymerase adds nucleotides to the primers to complete the synthesis of the new complementary strands. Slide5

These three steps- denaturation, annealing, and extension-constitute one PCR "cycle

".

This process is typically repeated for 30-40 cycles, amplifying the target sequence exponentially.Slide6

** When performing a PCR reaction for your DNA sample it is advised to perform 2 other reactions (Negative control , Positive control ) .Negative control :To check for contamination , contain all PCR components except the template replaced with water .

Positive control :

To check if PCR reaction has worked or not , contain all components and a known target to compare the unknown sample with it .Slide7

Experimental procedure :To prepare 20 μl PCR reaction mixture :

1. Mark 3 Sterile PCR tubes as ( Negative control , Positive control and sample

).

2

. Add (10 μl ) master mix , (2 μl ) Template DNA , (2 μl ) Forward Primer , (2 μl ) Reverse Primer to each tube

.

3. To the tube marked as Negative control add ( 2 μl ) Sterile deionized water , to the Positive control tube add ( 2 μl ) known DNA fragment , and to the Sample tube add ( 2 μl ) of your DNA sample

.

4. Complete the volume in each tube to (20 μl ) with Sterile deionized water.Slide8

5. Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube.

6

. Place the 3 tubes in PCR device and set up the PCR programme to start the process .

* Example of PCR program:

1. Initial denaturation at 94°C for 1 min

1

cycle.

2. Denaturation at 94°C for 30 sec

3. Annealing at 55



C for 30 sec.

4. Extension at 72°C for 1 min.

5. Repeat step 2-

35cycles

6. Final extension at 72°C for 2 min. 1 cycle

7. Hold at 4



C until ready to load onto gel.