1. Nested priming. This method was developed to normalize the PCR relative to mRNA abundance. If two different messages have equally good matches with the first arbitrary primer, but their abundance differs by 10-fold, the differentially displayed bands will differ in intensity by 10-fold. The introduction of a nested primer with a new overhanging nucleotide at the 3'-end may give a chance to the least abundant message to be amplified if it happens that the new nucleotide matches the sequence. Therefore, by adding one nucleotide at the 3' end, 1/16 of the previously amplified molecules will be further amplified. If two nucleotides overhang, then only 1/256 parts will be amplified and so forth. A practical example of nested oligonucleotide design and their use are extensively discussed in ref. 6.
2. For nested primer amplification, proceed as in cDNA synthesis. Subject the sample to amplification as described earlier except that only 10 high stringency cycles are to be performed. After this, transfer 3.5 pL of each reaction to tubes containing 36.5 pL of: dH2O, 22.0 pL, 10X second-strand buffer 4.0 pL, 10 pmol/mL nested primer 4.0 pL, 1 M DTT 4.0 pL, 1 mM dNTP 2.0 pL, Taq DNA polymerase 0.5 pL. Subject the sample to 40 high stringency cycles and procceed as above (Subheading 3.2.1., step 7).
Alternatively, shorter oligonucleotides could be used for cDNA production and amplification. If, for example, 10 mers are used, the temperature of the high-stringency cycles should be dropped to as low as 37°C, and a slope period of 15 s should be allowed for the 37-72°C transition to prevent denaturation of the primer from the template. Also, shorter primers will have the tendency to generate less products than longer primers. The use of Stoffel fragment for amplification instead of AmpliTaq DNA polymerase will help to minimize this problem (14). Except for these considerations, the protocol to follow is the same as that already described for longer primers.
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