SGER: Anonymous Rare-Cutter Restriction Fragments: A NovelSource of Genetic Markers for Population Biology

9728376麦克唐纳这项研究的目的是开发一种新的技术--匿名稀有切割器限制性片段(ARRFs)，用于分析遗传变异。ARRF技术将包括用两种稀有切酶消化基因组DNA，从许多大片段中分离出少数小片段，并将小片段分解在凝胶上。因为没有PCR扩增步骤，所以条带的强度将与片段的拷贝数成正比。因此，单拷贝核片段应该与多拷贝和细胞器片段区分开来，杂合子和纯合子应该区分开来。寄生虫或污染的DNA也应该很容易被识别出来。每一对稀有切割酶都有可能产生数十个独立的遗传标记，所有这些标记都可以同时进行检测。将这项技术应用于新物种所需的唯一开发步骤是找到一种合适的技术来制备基因组DNA，并确定哪一对酶能产生合适数量的片段。AARF技术在种群生物学中需要遗传标记的广泛研究中是有用的，包括基因流和种群结构、杂交、交配行为、自交率和QTL定位的研究。与目前用于分析核DNA变异的其他技术相比，AARF技术的这些属性是主要优势。ARRF技术将逐步发展，首先是细菌DNA(基因组大小约4M碱基对)，然后是酵母(11MBP)，然后是果蝇(100MBP)，然后是更大的后生动物(1000MBP)。随着基因组变得更大，每个单拷贝片段在总DNA中所占的比例变得更小，增加了技术挑战。计算表明，从基因组大小为数十亿个碱基对的生物中检测ARRF条带所需的DNA量相当大，接近实用性的上限，因此不能保证ARRF技术在所有生物上都是可行的。然而，如果这项技术奏效，它将是一个简单、丰富和强大的遗传标记来源，将对广泛的种群生物学家有用，并有可能用于动植物疾病和作物改良的研究。

9728376 McDonald The object of this SGER research is to develop a new technique, anonymous rare-cutter restriction fragments (ARRFs) for assaying genetic variation. The ARRF technique would consist of digesting genomic DNA with two rare-cutter restriction enzymes, isolating the few small fragments from the many large fragments, and resolving the small fragments on a gel. Because there is no PCR amplification step, the intensity of a band will be proportional to the number of copies of the fragment. Therefore, single-copy nuclear fragments should be distinguishable from multiple-copy and organellar fragments, and heterozygotes should be distinguishable from homozygotes. DNA from parasites or contamination should also be easily be identified. Each pair of rare-cutter restriction enzymes has the potential to yield dozens of independent genetic markers, all of which could be assayed simultaneously. The only development steps required to apply the technique to a new species will be finding a suitable technique for preparing genomic DNA and determining which pair of enzymes yield suitable numbers of fragments. The AARF technique would be useful in a wide variety of studies in population biology where genetic markers are needed, including studies of gene flow and population structure, hybridization, mating behavior, selfing rates, and QTL mapping. These attributes of the AARF technique are major advantages over other techniques currently used for assaying variation in nuclear DNA. The ARRF technique will be developed gradually, starting with bacterial DNA (genome size about 4M base pairs), then yeast (11Mbp), then Drosophila (100 Mbp), then larger Metazoans (1000+Mbp). As the genome size gets larger, each single-copy fragments becomes a smaller fraction of the total DNA, increasing the technical challenges. Calculations indicate that the amount of DNA required for detecting ARRF bands from an organism with a genome size in the billions of base pairs is fairly large, near the upper limit of p racticality, so there is no guarantee the ARRF technique will be feasible on all organisms. If the technique works, however, it will be a simple, rich, and robust source of genetic markers that will be useful to a broad range of population biologists, as well, potentially, in studies of animal and plant disease and crop improvement.