Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification
Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification
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DOI：
10.1093/nar/gnf056
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发表时间：
2002-06-15
影响因子：
14.9
通讯作者：
Pals, G
中科院分区：
文献类型：
--
作者：
Schouten, JP;McElgunn, CJ;Pals, G
我们描述了一种新的方法相对定量的40种不同的DNA序列在一个易于进行的反应，只需要20纳克的人DNA。这种多重连接依赖性探针扩增（MLPA）技术的应用包括检测人类BRCA 1、MSH 2和MLH 1基因中的外显子缺失和重复，检测三体如唐氏综合征，表征细胞系和肿瘤样品中的染色体畸变以及SNP/突变检测。通过MLPA的mRNA的相对定量将在别处描述。在MLPA中，不是样品核酸而是添加到样品中的探针被扩增和定量。通过PCR扩增探针取决于样品中探针靶序列的存在。每个探针由两个寡核苷酸组成，一个是合成的，一个是M13衍生的，它们与靶序列的相邻位点杂交。连接这种杂交的探针寡核苷酸，允许随后的扩增。所有连接的探针具有相同的末端序列，允许仅使用一个引物对同时进行PCR扩增。每个探针产生130和480 bp之间的独特大小的扩增产物。探针靶序列较小（50-70 nt）。连接反应的先决条件提供了区分单核苷酸差异的机会。
We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M 13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have Identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.