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

9510733 Densmore这项研究将评估一种使用蛋白质编码基因的cDNA片段作为(大部分未开发的)遗传变异衡量标准的方法，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序列(TAPS)的技术将使用12种不同的“锚定”引物(寡核苷酸dT12+2个其他脱氧碱基)，通过反转录成cDNA，基本上从总的聚A(())RNA中挽救整个mRNAs群体。然后利用聚合酶链式反应(PCR)从每个单链cDNA中合成单个双链cDNA片段，方法是使用一个短的随机扩增或共同序列引物，该片段带有用于拯救mRNAs亚群的相同锚定引物(32P标记)。这两个引物之间的聚合酶链式反应扩增产生每一个mRNA1个放射性标记的cDNA片断(只要有一个与短随机引物互补的cDNAs序列)。用特定的锚定引物回收不同的mRNAs，用聚合酶链式反应(PCR)扩增出对应于不同mRNAs的多个cDNAs片段，用凝胶电泳法和放射自显影技术进行检测。使用不同的短引物与相同的锚定引物进行额外的PCR反应将产生额外的mRNA特异的cDNA片段，而额外的锚定引物允许检测其他亚群的cDNA。这项研究将确定是否可以用TAPS技术证明个体、谱系或物种特异的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被称为转录扩增多态序列(TAP)的方法，用于分析蛋白质编码基因的DNA片段，作为(基本上未开发的)遗传变异的衡量标准，并将开始确定其在进化生物学中的应用范围。这项研究将确定是否可以通过TAPS技术证明遗传或个人、世系或物种特定的DNA片段，并将开始评估该方法在定量分析群体遗传变异方面的实用性。***

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. ***