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Sunday, March 17, 2019

How to prevent PCR amplicon contamination

PCR - Polymerase Chain Reaction is very sensitive technique for the amplification of Nucleic Acids. Due to its sensitive nature, billions of copies of DNA can be produced from tiny amount of initial DNA. If not properly handled this can lead to carry over contamination: from previously amplified PCR products.

To eliminate the issue of carry over contamination, most common method used to remove amplicon / PCR product is to use dUTP instead of dTTP and the use of an enzyme in the reaction mixture called the UNG, Uracil DNA Glycosylase.

Uracil DNA Glycosylase selectively removes dUTP incorporated PCR product,  without interfering with the target DNA amplification.

Uracil DNA Glycosylase:

Uracil DNA Glycosylase also known as UNG or UDG is an enzyme that degrades Uracil containing DNA.

Uracil-DNA Glycosylase can be used to cleave DNA at any site where a deoxyuridylate residue has been incorporated. The resulting abasic sites can then be hydrolyzed by:
  1. Alkali treatment
  2. High temperatures
  3. Endonucleases that cleave specifically at abasic sites.
Features of Uracil N Glycosylase
  1. The UNG enzyme is active only on Uracil containing DNA, inactive on RNA.
  2. Activity of the UNG enzyme is high on single stranded DNA(ssDNA) than double stranded DNA (dsDNA).
  3. Uracil DNA Glycosylase hydrolyzes the Uracil-glycosidic bonds at U-DNA sites in single stranded DNA and double stranded DNA, excising Uracil and creating alkali sensitive abasic sites in the DNA.
Mechanism of Action of Uracil DNA Glycosylase



Mechanism of Action of Uracil DNA Glycosylase

Applications of Uracil DNA Glycosylase:

  • The enzyme Uracil DNA Glycosylase can be used in site directed mutagenesis experiments to increase the efficiency.
  • UNG can be used in highly labelled oligo nucleotide probe production.
  • Uracil-DNA Glycosylase can be used with dUTP to eliminate PCR carryover contamination from previous DNA synthesis reactions.
Use of Uracil DNA Glycosylase for elimination of PCR product contamination:

Uracil DNA Glycosylase enzyme is very specific to Uracil containing DNA, cleaves uracil-glycosidic bonds there by degrading the DNA.

While setting up PCR reaction mixture, dUTP should be used instead of dTTP and UNG enzyme should be included in the PCR mixture.

Checking UNG Enzyme functionality by conventional PCR:

Generate PCR product using dUTP in the reaction mixture, Use a small amount of dUTP incorporated PCR product (10^5 fold diluted) as template for second round of PCR with UNG enzyme in the reaction mixture (follow intrusction guidelines by the manufacturer) and without UNG enzyme. Analyze the second round PCR product on Agarose gel.

Checking UNG Enzyme functionality by Real Time PCR:

Generate PCR product using dUTP in the reaction mixture, Use a small amount of dUTP incorporated PCR product (10^5 fold diluted) as template for second round of PCR with and ithout UNG enzyme in the reaction mixture (follow intrusction guidelines by the manufacturer).

Analyze and compare Ct values in the reaction mixture with and without UNG enzyme.

Types of Uracil DNA Glycosylase:

Uracil DNA Glycosylase from Ecoli s widely used in PCR, but this is not completely inactivated at higher temperatures, whereas Uracil DNA Glycosylase from Atlantic Cod is heat liable and this is suitable for 1 step RT-PCR.

Notes:
Use of UNG prevents future contamination. Existing contamination of DNA amplified with dTTP cannot be removed.