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RANDOM-BREAKAGE MAPPING METHOD APPLIED TO HUMAN DNA SEQUENCES

机译：适用于人类DNA序列的随机断裂映射方法

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摘要

The random-breakage mapping method [Game et al, (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts, The methodology involves Nod restriction endonuclease digestion of DNA from irradiated cells, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks, This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment, By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a Nod fragment on the X chromosome, thus demonstrating the validity of the technique, We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding Nod fragments on chromosome 21, A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed, Since both probes D21S1 and D21S11 hybridized to a single Nod fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.
机译：将随机断裂作图法[Game等，（1990）Nucleic Acids Res。，18，4453-4461]应用于人成纤维细胞中的DNA序列。该方法包括从照射的细胞中对DNA进行Nod限制性核酸内切酶消化，然后进行脉冲场凝胶电泳，Southern印迹以及与识别目标单拷贝序列的DNA探针杂交。 Southern印迹显示了完整的限制性片段的条带，并且由于辐射诱导的随机断裂而在该条带以下出现了条带。该条带斑在对应于杂交位点到限制性片段两端的距离的位置上包含两个强度上的不连续性通过分析这些不连续点的位置，我们确定了探针DXS1327在X染色体上Nod片段内的先前映射位置，从而证明了该技术的有效性。我们还能够相对于探针D21S1和D21S15进行定位。据报道，它们的相应Nod片段位于21号染色体的末端，第三个21号染色体探针D21S11接近D21S1，尽管存在第二个可能位置的不确定性，因为D21S1和D21S11探针均与单个Nod片段杂交并产生类似的拖影模式，这种不确定性可通过随机断裂映射方法消除。

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