9510733 Densmore This research will evaluate a method for using cDNA fragments from protein coding genes as a (largely untapped) measure of genetic variation, and will begin to determine the scope of its application to evolutionary biology. The technique, named Transcribed Amplified Polymorphic Sequences (TAPS), will use 12 different "anchoring" primers (oligo dT12 + 2 other deoxy bases) to rescue essentially the entire population of mRNAs from total poly A(() RNA by reverse transcription into cDNAs. The polymerase chain reaction (PCR) is then used to synthesize a single double-stranded cDNA fragment from each single-stranded cDNA by using a short randomly amplified or consensus sequence primer with the same anchoring primer (radiolabelled with 32p) used to rescue the subpopulation of mRNAs. PCR amplification between these two primers produces one radiolabelled cDNA fragment per mRNA (provided there is a cDNA sequence complementary to the short random primer). Multiple cDNA fragments, which correspond to different mRNAs that were rescued by a specific anchoring primer and amplified by PCR, are resolved by gel electrophoresis and detected by autoradiography. Additional PCR reactions using different short primers with the same anchoring primer will yield additional mRNA-specific cDNA fragments, while additional anchoring primers allow other subpopulation of cDNAs to be tested. This study will determine whether the inheritance of individual-, lineage-, or species-specific cDNA fragments can be demonstrated by the TAPS technique, and will begin to assess the utility of this method for quantitative analysis of genetic variation in populations. %%% This research will evaluate a method called Transcribed Amplified Polymorphic Sequences (TAPS) for analyzing DNA fragments from protein coding genes as a (largely untapped) measure of genetic variation, and will begin to determine the scope of its application to evolutionary biology. The study will determine whether the inheritance or individual- , lineage-, or species-specific DNA fragments can be demonstrated by the TAPS technique, and will begin to assess the utility of this method for quantitative analysis of genetic variation in populations. ***
