Baldocchi R A, Tartaglia K E, Bryda E C, Flaherty L
Molecular Genetics Program, Wadsworth Center, Albany, New York 12201, USA.
Genomics. 1996 Apr 15;33(2):193-8. doi: 10.1006/geno.1996.0183.
Representational difference analysis (RDA) is a subtractive hybridization technique by which the differences between two complex genomes can be isolated. An improved version of this technique was used to isolate DNA segments that map to a narrow genetic region adjacent to the jcpk locus on Chromosome 10 of the mouse. A mutation at this locus acts recessively and causes an early onset polycystic kidney disease. Genomic subtractions involving DNA from C57BL/6 (B6) and its partially congenic partner, B6-jcpk/jcpk, produced 39 restriction fragments (difference products), 25 of which were unique and represented differences in BglII sites between these two strains. Although none identified the jcpk locus itself, 7 of these were mapped to an interval between 3.4 and 6.5 cM distal to the jcpk locus. Five of these 7 difference products were developed by subtracting B6-jcpk/jcpk from B6 DNA, but only 1 of the 5 was isolated using the original RDA technique. The other 4 were obtained by an improved technique that included size selection of difference products after the third round of subtractive hybridization and amplification. The remaining 2 of the mapped products resulted from the reciprocal subtraction experiment using the improvements. Thus, by this improved technique and two-way subtraction, we were able to add seven new markers to a relatively small genetic region on Chromosome 10.
代表性差异分析（RDA）是一种消减杂交技术，通过该技术可以分离两个复杂基因组之间的差异。此技术的一个改进版本被用于分离定位到小鼠10号染色体上与jcpk基因座相邻的狭窄遗传区域的DNA片段。该基因座的突变呈隐性，会导致早发性多囊肾病。涉及C57BL/6（B6）及其部分同源伴侣B6-jcpk/jcpk的基因组消减产生了39个限制性片段（差异产物），其中25个是独特的，代表了这两个品系之间BglII位点的差异。尽管没有一个能鉴定出jcpk基因座本身，但其中7个被定位到jcpk基因座远端3.4至6.5 cM的区间。这7个差异产物中有5个是通过从B6 DNA中减去B6-jcpk/jcpk得到的，但这5个中只有1个是使用原始RDA技术分离出来的。另外4个是通过一种改进技术获得的，该技术包括在三轮消减杂交和扩增后对差异产物进行大小选择。其余2个定位产物来自使用这些改进方法的反向消减实验。因此，通过这种改进技术和双向消减，我们能够在10号染色体上一个相对较小的遗传区域添加7个新的标记。
