Is there any cross-reaction or mistake signal intensity?
What are the best techniques in comparison with Nested PCR?
Hello Hazem Almhanna
Is there any cross-reaction or mistake signal intensity?
Nested PCR employs two sets of primers and two successive PCR reactions. Although the nested PCR is one of the most sensitive PCR techniques, the possible cross-contaminations and carry over contaminations are the major difficulties associated with it. They may occur during opening of reaction tubes, during preparation for the second step amplification by transferring amplicons already produced during the first amplification step, subsequently resulting in false positives thereby reducing the accuracy of the test.
Thus, the increased sensitivity of nested PCR is bought at the price of potential false positives, because tubes containing high concentrations of the first amplification product must be opened and manipulated to set up the second amplification.
What are the best techniques in comparison with Nested PCR?
A method for efficient nested PCR that eliminates the need for these manipulations would reduce the chance for false positives. The approach described in the article attached below is based on the use of a single reaction tube that remains closed for both amplifications after reagents are introduced for each. Reagents for the second amplification are sequestered and preserved in the reaction tube during the first amplification, and then introduced into the reaction mixture for the second amplification.
Please refer to the method described in the paper attached below.
https://genome.cshlp.org/content/2/1/60.full.pdf+html
The strategy employed is provided below.
1. PCR amplification 1 with template DNA is set up in the usual fashion in reaction tubes and overlaid with mineral oil. Outer primers are used at reduced concentration.
2. Master mix for amplification 2 is prepared in a melted, thin agarose gel matrix and introduced into the lid lock chamber of the reaction tubes. This mix contains inner primers at severalfold excess over outer primers for amplification 1.
3. The tube dead space is filled with mineral oil to just below the lid lock line before tube closure.
4. While the first amplification is run, the master mix for the second remains sequestered at the tube top, protected from extreme temperature changes to which the lower end of the tube is subjected.
5. After the first amplification is completed, the reagents for the second amplification are introduced by brief centrifugation into the reaction space. The second amplification is then run as usual.
Hope this will help!
Good Luck!
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