Moir D T, Dorman T E, Xue F, Ma N S, Stanton V P, Housman D, Bowden D W, Noll W W, Mao J
Department of Human Genetics and Molecular Biology, Collaborative Research, Inc., Waltham, MA 02154.
Gene. 1993 Dec 22;136(1-2):177-83. doi: 10.1016/0378-1119(93)90461-b.
An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.
在酵母人工染色体（YAC）文库中进行的一系列基于杂交的染色体步移实验中，分离出了一组重叠的21个YAC，它们覆盖了人10号染色体上的RET原癌基因[高桥等人，《癌基因》3（1988）571 - 578]和D10S102标记。遗传连锁分析表明，这个染色体区域是负责2A型多发性内分泌肿瘤（MEN2A）的基因所在位置。使用携带RET序列标签位点（STS）的4个YAC和携带D10S102 STS的2个YAC来启动染色体步移。这些步移基于YAC的Alu元件介导的聚合酶链反应（Alu-PCR）产物与YAC克隆复杂文库的Alu-PCR产物点杂交。通过对单个YAC的Alu-PCR产物印迹进行探针杂交以及比较每个YAC的Alu-PCR指纹图谱，确认了步移中鉴定出的YAC的杂交锚定内容。在由21个重叠YAC形成的11个区间内，对10个基于杂交的Alu-PCR锚定和3个STS锚定进行了排序。YAC中需要最少间隙的锚定顺序与步移结果一致，并确定STS锚定顺序为D10S102 - D10S94 - RET。根据重叠群中4个代表性YAC的限制性图谱，这组重叠的YAC代表了约1.55 Mb的人类基因组。这些结果证明了Alu-PCR杂交用于染色体步移的强大功能，并提供了丰富的重叠YAC来源，可用于鉴定候选的MEN2A基因。
