PCR Notes

Polymerase Chain Reaction (PCR)

Overview

PCR is a method used to amplify a specific DNA sequence, creating millions or billions of identical copies. This process is quicker, more efficient, and more accurate than older methods like integrating a gene into a bacterial plasmid.

Ingredients for PCR

To carry out a successful PCR, you need four main ingredients:

  • Target DNA molecule: The double-stranded DNA segment containing the gene or sequence you want to amplify.

  • DNA primers: Short sequences of DNA (20-30 nucleotides) that initiate DNA synthesis.

  • Heat-resistant DNA polymerase: An enzyme that replicates DNA at high temperatures without denaturing.

  • Deoxyribonucleotide triphosphates (dNTPs): The building blocks of DNA (adenine, guanine, cytosine, and thymine).

Steps in a PCR Cycle

A single PCR cycle consists of three steps:

  1. DNA Strand Separation (Denaturation)

  2. Primer Annealing (Hybridization)

  3. DNA Synthesis (Elongation)

Step 1: DNA Strand Separation (Denaturation)

  • Increase the temperature of the solution to about 95^{\circ}C for 15 seconds.

  • This breaks the hydrogen bonds between the two DNA strands, separating them.

  • The target sequence is flanked by flanking sequences, where the primers will bind.

Step 2: Primer Annealing (Hybridization)

  • Cool the solution to about 54^{\circ}C.

  • At this temperature, DNA primers bind to the flanking sequences.

  • Primers are complementary to the flanking sequences.

  • One primer binds to the 3' end of one strand, and the other binds to the 3' end of the complementary strand.

  • The DNA polymerase can only elongate the DNA in the 5' to 3' direction, so the primers must bind accordingly.

Step 3: DNA Synthesis (Elongation)

  • Increase the temperature to about 72^{\circ}C.

  • This is the optimal temperature for the heat-resistant DNA polymerase.

  • The DNA polymerase binds to the DNA and adds deoxyribonucleotide triphosphates, elongating and synthesizing new DNA strands.

  • At the end of this step, two identical copies of the original DNA segment have been formed.

Amplification Over Multiple Cycles

Each PCR cycle doubles the amount of DNA. After one cycle, one becomes two.

The equation that gives us the total number of copies made after n cycles is:

2^n

Cycle Number

Number of Copies

1

2

2

4

3

8

4

16

PCR Cycles

About 20-30 cycles can occur in an hour, resulting in millions to billions of copies of the DNA segment. PCR is therefore a very effective and efficient method for amplifying DNA.

An important property of PCR is that it occurs within a closed container. After the first cycle, nothing needs to be added to the mixture, as it already contains all the necessary ingredients. To restart the cycle, the temperature is increased to 95^{\circ}C.

PCR Steps

  1. Increase temperature to 95^{\circ}C to separate the two DNA strands.

  2. Cool the solution to 54^{\circ}C.

  3. Increase the temperature to 72^{\circ}C to allow for DNA synthesis.

PCR is easy to control because it is performed in a closed container with all the necessary ingredients. The temperature is simply changed to ensure that the cycles continue.

PCR is a more effective method than using bacterial cells to amplify genes of interest.