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applexixixi

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[交流] 想扩增10kb多的基因簇，现在有什么技术可以一次性扩增出来吗

10kb多的基因簇是连续的，想扩增出来，在此问问虫友们现在有什么技术可以解决这个问题呢？

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1楼 2012-10-24 16:40:25

已阅回复此楼关注TA 给TA发消息送TA红花 TA的回帖

huiee

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applexixixi(金币+1): 谢谢参与
小丹木木: 金币+2, 鼓励交流 2012-10-27 13:46:40

推荐一篇paper 如果愿意用的话
Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

Jun Fu,
Xiaoying Bian,
Shengbaio Hu,
Hailong Wang,
Fan Huang,
Philipp M Seibert,
Alberto Plaza,
Liqiu Xia,
Rolf Müller,
A Francis Stewart
& Youming Zhang

Affiliations
Contributions
Corresponding authors

Nature Biotechnology
30,
440–446
(2012)
doi:10.1038/nbt.2183

Received
24 October 2011
Accepted
16 March 2012
Published online
29 April 2012

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.