Unit 1: Polymerase Chain Reaction

Introduction

Polymerase chain reaction (PCR) is an artificial in vivo (outside of the cell) process simulating DNA replication of a selected segment, allowing the selection and rapid amplification of a DNA fragment. The PCR machine is a thermal cycler which controls the temperature inside the container to a very accurate scale, facilitating PCR.

Reaction Mixture

Reaction mixture is the aqueous solution containing all the necessary ingredients (eg. DNA, dNTPs, Buffer, Primer and DNA Polymerase) , in performing PCR. It will be directly heated and cooled in the PCR thermal cycler, which allows for the denaturation (separation) of DNA strands, annealing of primer and extension of dNTPs.

  1. DNA sample
  2. Original un-selected double-stranded template DNA
  3. dNTPs
  4. Deoxynucleotides, which is the monomer of DNA molecules. Free-floating nitrogen bases. dNTPs bind to single-stranded DNA during DNA synthesis.
  5. Taq polymerase
  6. The denaturing of DNA requires a high temperature of 95℃. Normal enzymes denature at such high temperature. Taq polymerase is isolated from a heat-tolerant bacterium. To prevent the denaturing of DNA polymerase, Taq polymerase is used.
  7. Primers
  8. Primers are single-stranded short sequences of DNA (usually 18-22 bp) acting as the starting points for DNA synthesis. Two primers are used in each PCR reaction. They are designed so that they flank the target region (region to be amplified). The design will be discussed later.
  9. Buffer
  10. DNA is sensitive to pH. At low pH levels (< 7), the chemical bonds that bind nucleobases to DNA are broken by hydrolysis. At higher pH levels (> 10), deprotonation disrupts the dinucleotide bonds that form the double helix. Buffer resists pH changes by chemically neutralizing small amounts of added acidic or basic compounds, thus maintaining the overall pH of a medium.

Steps of PCR

  1. DNA Denaturation
    • 95℃
    • The 2 DNA strands separates
  2. Annealing of primers
    • 65℃
    • primers bind to single-stranded template DNA complementarily at the edges of the target region
  3. DNA synthesis (primer extention)
    • 70℃
    • Taq polymerase catalyse the addition of dNTPs

(3’ to 5’)

4. Repeating the cycle

    • Amout of DNA is doubled in each cycle
    • generally, the cycle is repeated ~40 times

Designing Primers

The lengths of primers are usually 18-22 bp. The primers bind to the template by complementary base pairing. The new strand of DNA is produced from 3’ to 5’ of the template strand. Therefore,

Design of Primer 1: forward, complimentary to start of gene

Design of Primer 2: reverse, end of gene

If you wish to know more about how to maximizing the yield of PCR by optimizing the primers, you can read

Alternatively, you may choose to use PCR primer designing tools to aid your design. This is a recommendation of such tool,

Applications

  • Analyzation

Sample for gel electrophoresis or sequencing

  • Detection of mutation

Design the primer to bind to a specific mutated sequence. Then run gel to check if the DNA has that sequence.

For example, anemia involves a mutation in the haemoglobin gene. PCR primers are designed to bind to the mutated sequence. Therefore, after amplification, mutated gene is shorter than the normal gene. By comparing the mutated and normal gene by gel electrophoresis, the mutated DNA is further from the well than the normal gene.

Detection of mutation in a gene by cleavage location

http://www.jlr.org/content/39/5/1101/F1.expansion

  • Cloning

Amplification of DNA increases the successful rate and yield of restriction ligation.