Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

被引:57
作者
Kouprina, N
Annab, L
Graves, J
Afshari, C
Barrett, JC
Resnick, MA
Larionov, V
机构
[1] NIEHS, Mol Genet Lab, NIH, Res Triangle Pk, NC 27709 USA
[2] NIEHS, Mol Carcinogenesis Lab, NIH, Res Triangle Pk, NC 27709 USA
关键词
gene isolation; yeast artificial chromosome;
D O I
10.1073/pnas.95.8.4469
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Unique, small sequences (sequence tag sites) have been identified at the 3' ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3' unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3' hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3' end sequence to various Alu positions as much as 600 kb upstream, These YACs were retrofitted with a Neo(R) and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.
引用
收藏
页码:4469 / 4474
页数:6
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