Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers
Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers
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DOI：
10.1006/geno.1995.1264
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发表时间：
1995-12-10
期刊：
影响因子：
4.4
通讯作者：
King, RA
中科院分区：
文献类型：
--
作者：
Oetting, WS;Lee, HK;King, RA
由于短串联重复多态性（STRPs）在人类基因组中数量众多且具有高度多态性，因此将其作为连锁分析的标记位点变得越来越重要。使用自动DNA测序仪对STRP模式进行基于荧光的检测，通过消除对放射性的需要和产生可用于计算机分析的数字化自射线图样图像，提高了该技术的效率。为了简化程序并降低荧光STRP分析的成本，我们开发了一种称为带尾引物（MSTP）的多路STRP技术，该技术使用的引物具有19 bp的延伸，与M13测序引物的序列相同，在前引物的5'端与多路引物对一起进行单次聚合酶链反应（PCR）扩增。通过添加M13引物-染料共轭物作为与荧光染料共轭的唯一引物来检测条带模式，从而消除了将红外荧光染料与STRP引物直接共轭的需要。使用MSTP进行连锁分析大大减少了PCR反应的数量。在同一反应中，最多可以有5对引物组合在一起。目前，在整个常染色体基因组中，平均遗传距离为28 cM的一组148个STRP标记可通过37组多重扩增反应进行分析。我们已经自动分析了这些模式的链接使用软件，既检测STRP带模式，并确定其大小。然后可以从数据库管理器以用户定义的格式导出此信息，以便进行链接分析。(C) 1995学术出版社，Inc。
The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) using primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5' end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye to the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis. (C) 1995 Academic Press, Inc.