Exon mapping by PCR.
Exon mapping by PCR.
复制标题
通过 PCR 进行外显子作图。
DOI：
10.1093/nar/21.3.769
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发表时间：
1993
影响因子：
14.9
通讯作者：
Crouse,GF
中科院分区：
文献类型：
--
作者：
Niu,L;Crouse,GF
一旦知道了信使核糖核酸的cdna序列和该基因的基因组克隆，就很容易找到该基因的外显子并对其进行排序。人们可以根据cdna序列制作寡核苷酸引物，并使用这些引物寻找包含外显子序列的基因的亚克隆。然后可以用相同的寡核苷酸来确定该外显子的一部分的序列。然而，如果不对整个基因进行测序，就很难确定外显子的准确位置。我们提出了一种在周围DNA序列未知的情况下确定外显子准确位置的快速方法。我们最近已经对小鼠Rep-3基因的全部26个外显子进行了测序[(1)；LN和GFC，手稿正在准备中]。这些外显子分布在134kb的DNA上，由于该基因的大小，只对外显子和两侧的外显子/内含子连接进行了测序。亚克隆该基因的含有外显子的片段(通过与cDNAs或寡核苷酸杂交确定)，然后使用来自该cDNAs序列的寡核苷酸引物进行测序。该方法给出了外显子一侧的外显子/内含子边界的序列；合成了另一条外显子/内含子边界的序列，以获得另一条连接序列。虽然这种方法可以相对快速地分析外显子序列，但我们没有获得外显子在克隆DNA片段中的位置的信息。然而，所有的基因组片段都被克隆到了Bluescrip载体(Stratagene)中，并且证明了通过使用基因特异性测序引物和来自载体的一条引物通过PCR获得从外显子到限制片段边界的距离是可能的，如图1所示。这种方法有几个优点：(1)由于来自载体的引物已经可用，所以不需要合成其他引物，因为它们是用于对Bluescrip载体中的插入片段进行测序的相同的引物。(2)不需要知道外显子在插入片段中的位置；两个单独的聚合酶链式反应可以用测序引物和每个侧翼引物来进行。只有正确定位的PCR反应才会产生产物，所以这种方法实际上可以确定限制片段内外显子的方向。(3)除了非常大的亚克隆外，该方法对所获得的值进行了内部检查，因为通过使用每个带有适当载体引物的测序引物可以获得外显子到两个载体边界的距离。(4)如果限制片段包含两个或更多外显子，则可以使用来自每个外显子的测序引物来确定这些外显子之间的距离。(5)该方法给出了从外显子到限制的距离
Once the cDNA sequence of a mRNA is known and genomic clones of the gene are available, it becomes a fairly easy task to find and sequence the exons of the gene. One can make oligonucleotide primers based on the cDNA sequence and use those primers to find subclones of the gene containing exon sequences. The sequence of a portion of the exon can then be determined with the same oligonucleotide. However, if the entire gene is not to be sequenced, it can prove difficult to determine the precise location of the exon. We present a quick method for determining the precise location of an exon when the sequence of the surrounding DNA is not known. We have recently sequenced all 26 exons of the mouse Rep-3 gene [(1); LN and GFC, manuscript in preparation]. These exons are spread over 134 kb of DNA and because of the large size of the gene, only the exons and the flanking exon/intron junctions were sequenced. Exon-containing fragments of the gene (determined by hybridization with either cDNA or oligonucleotides) were subcloned and then sequenced using an oligonucleotide primer derived from the cDNA sequence. This primer gave the sequence of the exon/intron boundary on one side of the exon; another primer was synthesized to enable sequencing in the opposite direction in order to obtain the other junction sequence.Although this method allowed a relatively rapid analysis of the exon sequences, we obtained no information about the location of the exon within the cloned DNA fragment. However, all of the genomic fragments had been cloned into Bluescript vectors (Stratagene) and it proved possible to obtain the distance from the exon to the boundary of the restriction fragment by PCR using the gene specific sequencing primer and a primer from the vector, as illustrated inFigure 1. There are several advantages of this method:(1) No other primers had to be synthesized since the primers from the vector were already available, as they are the same primers used for sequencing inserts in the Bluescript vectors.(2) It is not necessary to know the orientation of the exon within the insert; two separate PCR reactions can be done with the sequencing primer and each of the flanking primers. Only a PCRreaction that is correctly oriented will generate a product, so this method can in fact determine orientation of the exon within the restrictionfragment.(3) Except for extremely large subclones, the method has an internal check on the value obtained since the distance of the exon to both of the vector boundaries can be obtainedby using each of the sequencing primers with the appropriate vector primer.(4) If a restriction fragment contains two or more exons, the distance between those exons can be determined using a sequencing primer from each.(5) This methodgives the distance from the exon to the restriction