Testing a Newly Developed Molecular Genetic Methodology for use in Comparative Biology: TAPS

9510733 Densmore本研究将评估一种利用蛋白质编码基因的cDNA片段作为遗传变异（很大程度上尚未开发）测量的方法，并将开始确定其在进化生物学中的应用范围。这项名为转录扩增多态性序列（TAPS）的技术将使用12种不同的“锚定”引物（oligo dT12 + 2个其他脱氧碱基），通过反转录将整个mrna群体从总聚A(() RNA中拯救出来。然后，聚合酶链反应（PCR）通过随机扩增的短引物或具有相同锚定引物（用32p放射性标记）的一致序列引物，从每个单链cDNA合成单个双链cDNA片段，用于拯救mrna亚群。这两个引物之间的PCR扩增每个mRNA产生一个放射性标记的cDNA片段（前提是有一个cDNA序列与短随机引物互补）。多个cDNA片段对应于不同的mrna，这些mrna由特定的锚定引物拯救并通过PCR扩增，通过凝胶电泳分离并通过放射自显影检测。使用不同的短引物和相同的锚定引物进行额外的PCR反应将产生额外的mrna特异性cDNA片段，而额外的锚定引物允许测试其他cDNA亚群。本研究将确定是否可以通过TAPS技术证明个体、谱系或物种特异性cDNA片段的遗传，并将开始评估该方法在群体遗传变异定量分析中的实用性。这项研究将评估一种称为转录扩增多态性序列（TAPS）的方法，该方法用于分析蛋白质编码基因的DNA片段，作为一种（很大程度上尚未开发的）遗传变异测量方法，并将开始确定其在进化生物学中的应用范围。该研究将确定遗传或个体、谱系或物种特异性DNA片段是否可以通过TAPS技术证明，并将开始评估该方法在种群遗传变异定量分析中的效用。＊＊＊

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. ***