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Published on: May 22, 2012
Decontaminating the polymerase chain reaction
1Laboratory of Viral Diseases NIAID, NIH, Bethesda, MD 20892.
Biotechniques
|January 1, 1991
Summary
Restriction enzymes effectively inactivate contaminating DNA in polymerase chain reactions. This method ensures accurate PCR results by removing unwanted template DNA before amplification.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Polymerase chain reaction (PCR) is susceptible to contamination from template DNA.
- Unwanted template DNA can lead to false positive results in molecular biology applications.
- Effective methods are needed to eliminate contaminating DNA in PCR reactions.
Purpose of the Study:
- To investigate the efficacy of restriction enzyme digestion in inactivating template DNA for PCR.
- To demonstrate a method for preventing DNA contamination in PCR experiments.
Main Methods:
- Simulated DNA contamination by adding template DNA to a modified PCR mixture.
- Inactivated template DNA using restriction enzyme digestion with enzymes recognizing four-base sequences.
- Performed PCR amplification after enzyme inactivation and addition of fresh reagents.
Main Results:
- Restriction enzyme digestion successfully inactivated the added template DNA.
- PCR proceeded accurately after the removal of contaminating DNA.
- Demonstrated effectiveness with various restriction enzymes targeting short recognition sites.
Conclusions:
- Restriction enzyme digestion is a viable strategy to eliminate contaminating template DNA in PCR.
- This method enhances the reliability of PCR results by preventing false positives.
- Further research can explore enzyme selection for different DNA sequences and applications.
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