生物技术通报
生物技術通報
생물기술통보
BIOTECHNOLOGY BULLETIN
2010年
4期
156-160
,共5页
雷小春%司苏晋%薛亮亮%刘田福
雷小春%司囌晉%薛亮亮%劉田福
뢰소춘%사소진%설량량%류전복
重叠延伸PCR%甘丙肽%内含子%重构分子
重疊延伸PCR%甘丙肽%內含子%重構分子
중첩연신PCR%감병태%내함자%중구분자
Overlap extention%PCR%Galanin%Intron%Reconstructed molecule
构建嵌入第二内含子的甘丙肽(Galanin,GAL)全长基因组cDNA的重构分子.通过RT-PCR扩增出cDNA编区的序列,分别从基因组中扩增出cDNA的5'和3'端部分非编码序列;使用重叠延伸PCR(overlap extention PCR,OE-PCR)方法将三个片段重叠获得全长cDNA序列;再将全长cDNA从第三外显子第15个碱基处分成两部分,分开的cDNA前半部分和后半部分以及第二内含子进行重叠延伸获得重构分子,含有第二内含子的甘丙肽(GAL)全长基因组cDNA;将重构分子连入pMDI9-T simple载体.电泳分析观察到清晰的重构分子片段;测序显示重构分子由所设计的序列组成,第二内含子插入的位置准确,且无移码.使用重叠延伸PCR能够成功在cDNA中插入内含子获得一段重构基因.
構建嵌入第二內含子的甘丙肽(Galanin,GAL)全長基因組cDNA的重構分子.通過RT-PCR擴增齣cDNA編區的序列,分彆從基因組中擴增齣cDNA的5'和3'耑部分非編碼序列;使用重疊延伸PCR(overlap extention PCR,OE-PCR)方法將三箇片段重疊穫得全長cDNA序列;再將全長cDNA從第三外顯子第15箇堿基處分成兩部分,分開的cDNA前半部分和後半部分以及第二內含子進行重疊延伸穫得重構分子,含有第二內含子的甘丙肽(GAL)全長基因組cDNA;將重構分子連入pMDI9-T simple載體.電泳分析觀察到清晰的重構分子片段;測序顯示重構分子由所設計的序列組成,第二內含子插入的位置準確,且無移碼.使用重疊延伸PCR能夠成功在cDNA中插入內含子穫得一段重構基因.
구건감입제이내함자적감병태(Galanin,GAL)전장기인조cDNA적중구분자.통과RT-PCR확증출cDNA편구적서렬,분별종기인조중확증출cDNA적5'화3'단부분비편마서렬;사용중첩연신PCR(overlap extention PCR,OE-PCR)방법장삼개편단중첩획득전장cDNA서렬;재장전장cDNA종제삼외현자제15개감기처분성량부분,분개적cDNA전반부분화후반부분이급제이내함자진행중첩연신획득중구분자,함유제이내함자적감병태(GAL)전장기인조cDNA;장중구분자련입pMDI9-T simple재체.전영분석관찰도청석적중구분자편단;측서현시중구분자유소설계적서렬조성,제이내함자삽입적위치준학,차무이마.사용중첩연신PCR능구성공재cDNA중삽입내함자획득일단중구기인.
The study was design to construct a recombinant gene composed of galanin(GAL)full-length cDNA and the second intron of the galanin gene.Firstly,by RT-PCR the coding cDNA sequence was amplified,and the 5' and 3' noncoding sequences of Gal cDNA was amplified by PCR.Secondly,the full-length cDNA sequence was obtained by overlap extention PCR(OE-PCR).Thirdly,the full-length cDNA was cuted into two part at the 15 bp of the third exon and amplified them respectively.Finally,Using the two part of the full-length cDNA and the second intron as a template by OE-PCR,and the second intron was inserted into the galanin full-length cDNA,and the recombinant molecule was obtained then cloned into pMD19-T simple vector.The electrophoresis analysis illustrated a clear band of recombinant molecule fragment,and the squencing result proved that the recombinant molecule was composed of the second intron and full-length cDNA of galanin.The insertion position of the second intron is accurate without base malposition.Therefore,it proved that the Gal recombinant gene was obtained successfully by OE-PCR.