使用PCR辅助亲和色谱法从荧光原位杂交（FISH）探针中去除重复序列。

Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography.

作者信息

Craig J M, Kraus J, Cremer T

机构信息

Institute of Anthropology and Human Genetics, University of Munich, Germany.

出版信息

Hum Genet. 1997 Sep;100(3-4):472-6. doi: 10.1007/s004390050536.

DOI:10.1007/s004390050536

PMID:9272175

Abstract

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA.

摘要

荧光原位杂交（FISH）中使用的绝大多数探针都含有重复DNA。通常通过添加未标记的封闭剂Cot-1 DNA，使这种DNA在杂交反应中被竞争掉。我们已成功从两个复杂的FISH探针组中去除了重复DNA：一个简并寡核苷酸引物聚合酶链反应（DOP-PCR）单人类染色体文库和基因组DNA。该过程包括在溶液中使一个可通过DOP-PCR扩增的探针组与50倍过量的生物素标记的Cot-1 DNA杂交，并用链霉亲和素磁性颗粒捕获含Cot-1 DNA的杂交体，随后对未结合部分进行纯化和再扩增。通过与不含Cot-1 DNA的染色体杂交来检查探针的重复序列是否缺失。结果显示杂交模式与用未处理的探针与Cot-1 DNA杂交所获得的模式相当。