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Advances in quantitative PCR technology: 5' nuclease assays
1ViroLogic, Inc, South San Francisco, California, USA.
Current Opinion in Biotechnology
|March 21, 1998
Summary
5' nuclease assays offer a significant advancement in nucleic acid quantitation. This method uses Thermus aquaticus (Taq) polymerase exonuclease activity for precise DNA copy number measurement.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Nucleic acid quantitation is crucial in molecular biology.
- Traditional methods can be time-consuming and less sensitive.
Purpose of the Study:
- To introduce and explain the principles of 5' nuclease assays.
- To highlight their utility in nucleic acid quantitation.
Main Methods:
- Utilizing the 5'-3' exonuclease activity of Thermus aquaticus (Taq) polymerase.
- Employing dual-labelled probes that release a fluorogenic tag upon cleavage.
- Measuring fluorescence intensity either post-amplification or in real-time during PCR.
Main Results:
- The released fluorescence is directly proportional to the target nucleic acid concentration (copy number).
- Assays can be performed at endpoint or in real-time for dynamic monitoring.
- This method provides a sensitive and accurate means of quantitation.
Conclusions:
- 5' nuclease assays represent a significant advancement in nucleic acid quantitation.
- The real-time capability allows for efficient and precise monitoring of amplification.
- This technique is valuable for various applications requiring accurate DNA quantification.