Question

Description

The three steps—the separation of the strands, annealing the primer to the template, and the synthesis of new strands—take less than two minutes. Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been duplicated. The cycle can be repeated 30 or more times, and each newly synthesized DNA piece acts as a new template. After 30 cycles, 1million copies of the initial DNA piece can be produced

Question1

List all of the components of the “MASTER MIX” and describe their role in PCR.

Question2

Describe the contents of the two controls used in this procedure and their role in the process.

Question3

What is happening in the reaction during each of the following steps:

a. MELT:

b. ANNEAL:

c. EXTEND:

Question4

According to the output from the reaction, we could theoretically have over 1,000,000 copies of the PCR product from this reaction. What EXACTLY do we have 1,000,000 copies of?

Answer 1

Question 1)

There are following component in PCR:

* Taq Polymerase: this DNA polymerase is isolated from Thermus aquaticus bacteria. It has ability to synthesise DNA at high temperature.

* Magnesium containing buffer: Buffer provide suitable conditions for PCR working. Magnesium provide activity to DNA polymerase.

* Nucleotide (A, T, G and C) : these are the monomer sub unit in synthesis of DNA.

* Forward primer and Reverse primer: This primers have complementary base pair with DNA and provide 3' OH for activity of DNA polymerase.

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Question 2)

There are two types of control:

* positive control: This control is used to check that whether the components of PCR are working properly or not.

In this, we have added ultra pure DNA are used.

* Negative control: This is used to check whether there is contamination or not.

In this case either template or primer is not added. If there is band in gel appear. This shows contamination.

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Question 3)

melt: two DNA strand denaturated

Anneal: Primers are bound to the denatured strand of DNA.

Extend: DNA polymerase synthesise DNA on primers.

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Question 4)

We have 1,000,000 copies of newly synthesised fragment of DNA. This fragment is synthesised between the forward and reverse primer region of DNA.

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