High capacity thermal cycler for molecular genotyping

带有隐性热头(Hth)基因突变等位基因的拟南芥植株可以遗传其双亲染色体基因组中没有的祖先DNA序列信息。这种现象是全基因组的，在基因组的编码区和非编码区都会发生。这些发现完全是新奇的，提出的机制也是史无前例的。但这一发现在很大程度上是由于分子DNA技术的进步。随着聚合酶链式反应的出现，拟南芥基因组序列的完成以及寡核苷酸合成程序的进步，现在快速而可靠地对大量个体进行基因分型是微不足道的。因为这些分子标记是共显性的，所以也有可能同时对两条染色体进行采样。因此，识别携带两个等位基因的个体是非常简单的，或者是两个不同的等位基因在序列上只有一个核苷酸不同。在这项应用中，需要购买一台高容量的模块化热循环仪，用于对突变的红头病植物及其后代的群体进行基因分型。我建议使用聚合酶链式反应技术和分子标记来检测基因组编码区和非编码区DNA序列的变化。我们将使用这些简单的分析来阐明在这些突变体中发现的新型遗传不稳定的显著特征。如果在植物界以外的生物体中发现这一过程，这项研究可能会在未来具有重大的医学意义。

Arabidopsis thaliana plants homozygous for recessive mutant alleles of the hothead (hth) gene can inherit ancestral DNA sequence information that was not present in the chromosomal genome of their parents. This phenomenon is genome-wide and occurs in both coding and non-coding regions of the genome. These findings are completely novel and the mechanism proposed, unprecedented.  This discovery was made possible in large part by advances in molecular DNA technology. With the advent of PCR, the completion of the Arabidopsis genome sequence and advances in oligonucleotide synthesis procedures, it is now trivial to genotype large numbers of individuals rapidly and reliably.  Because these molecular markers are co-dominant it is also possible to sample both chromosomes simultaneous.  Hence, identifying individuals that carry two alleles that are identical or two distinct alleles that differ in sequence by only one nucleotide is very straightforward. In this application funds are requested to purchase a high capacity modular thermal cycler for genotyping populations of mutant hothead plants and their descendents.  I propose to use PCR technology and molecular markers to assay for changes in DNA sequences  in coding as well as  in non-coding regions of the genome. We will use these simple assays to elucidate the salient features of the novel type of genetic instability uncovered in these mutants.  If this process is found in organisms outside of the plant kingdom, it is possible that this research will have significant future medical relevance.