Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization
Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization
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DOI：
10.1101/gr.171701
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发表时间：
2001-06-01
期刊：
影响因子：
7
通讯作者：
Knoll, JHM
中科院分区：
文献类型：
--
作者：
Rogan, PK;Cazcarro, PM;Knoll, JHM
染色体重排经常通过荧光原位杂交（FISH）来监测，所述荧光原位杂交使用由通常远离疾病位点的连续基因组间隔组成的大型重组DNA探针。我们直接从人类基因组序列中开发了更小的、有针对性的单拷贝探针。这些单拷贝FISH（scFISH）探针通过类似于100-kb基因组序列的计算序列分析来设计。ScFISH探针通过长PCR产生，然后纯化、标记并单独或组合地与人类染色体杂交。由于scFISH探针缺乏重复的DNA序列，因此不需要用未标记的重复DNA进行预退火或阻断。杂交结果类似于常规FISH，除了较短的探针可以容易地可视化。来自同一区域的探针组合在中期染色体上产生单一杂交信号。ScFISH探针直接从基因组DNA产生，因此比重组DNA技术更快。我们开发了三个染色体区域的CDC 2L 1（染色体1 p36），MAGEL 2（染色体15q11.2），和HIRA（染色体22q11.2）基因的单拷贝探针，并显示其实用FISH。所测试的最小探针长度为2290 bp。为了评估scFISH对于高分辨率分析的潜在效用，我们确定了此类探针的染色体分布。这个长度或更长的单拷贝间隔在21号和22号染色体上分别平均间隔29.2和22.3 kb。这表明在中期染色体上观察到的异常可以用scFISH探针以高于先前可能的分辨率进行表征。
Chromosomal rearrangements are frequently monitored by Fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of contiguous genomic intervals that are often distant From disease loci. We developed smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computational sequence analysis of similar to 100-kb genomic sequences. ScFISH probes are produced by long PCR, then purified, labeled, and hybridized individually or in combination to human chromosomes. Preannealing or blocking with unlabeled, repetitive DNA is unnecessary, as scFISH probes lack repetitive DNA sequences. The hybridization results are analogous to conventional FISH, except that shorter probes can be readily visualized. Combinations of probes from the same region gave single hybridization signals on metaphase chromosomes. ScFISH probes are produced directly from genomic DNA, and thus more quickly than by recombinant DNA techniques. We developed single-copy probes for three chromosomal regions-the CDC2L1 (chromosome 1p36), MAGEL2 (chromosome 15q11.2), and HIRA (chromosome 22q11.2) genes-and show their utility for FISH. The smallest probe tested was 2290 bp in length. To assess the potential utility of scFISH For high-resolution analysis, we determined chromosomal distributions of such probes. Single-copy intervals of this length or greater are separated by an average of 29.2 and 22.3 kb on chromosomes 21 and 22, respectively. This indicates that abnormalities seen on metaphase chromosomes could be characterized with scFISH probes at a resolution greater than previously possible.