PCR with end trimming and cassette ligation: a rapid method to clone exon-intron boundaries and a 5'-upstream sequence of genomic DNA based on a cDNA sequence.
PCR with end trimming and cassette ligation: a rapid method to clone exon-intron boundaries and a 5'-upstream sequence of genomic DNA based on a cDNA sequence.
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DOI：
10.1101/gr.4.1.19
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发表时间：
1994-08
期刊：
影响因子：
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通讯作者：
H. Iwahana;Toshiyuki Tsujisawa;R. Katashima;Katsuhiko Yoshimoto;Mitsuo Itakura
中科院分区：
文献类型：
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作者：
H. Iwahana;Toshiyuki Tsujisawa;R. Katashima;Katsuhiko Yoshimoto;Mitsuo Itakura
我们描述了一种PCR扩增未知侧翼基因组DNA片段的方法。该方法是PCR与“末端修剪法”和“盒和盒引物法”相结合的方法。在该方法中，基因组DNA用三组不同的限制性内切酶消化。用BamHI、BglII、FbaI或MboI消化组1中的DNA。组2中的DNA用BlnI、NheI、SpeI或XbaI消化。第3组用SalI或XhoI酶切DNA。在仅存在一种dNTP的情况下，用DNA聚合酶I的Klenow片段对每组中消化的DNA进行末端修剪;对于组1、2和3，分别为dGTP、dCTP和dTTP。合成的盒C1、C2和C3分别具有5 ′-ATC-3 ′、5 ′-TAG-3 ′和5 ′-CGA-3 ′的5 ′突出序列。将每个相容盒分别连接到组1-3中的末端修剪的DNA。然后使用末端修剪和盒连接的DNA作为模板进行巢式PCR。与已知序列和盒退火的引物用于巢式PCR。将扩增的DNA片段在聚丙烯酰胺凝胶上电泳并纯化。将DNA片段克隆到pBluescript中后测定其序列。
We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-trimmed and cassette-ligated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript.