Mutation detection using Surveyor™ nuclease

被引:224
作者
Qiu, P [1 ]
Shandilya, H [1 ]
D'Alessio, JM [1 ]
O'Connor, K [1 ]
Durocher, J [1 ]
Gerard, GF [1 ]
机构
[1] Transgenom Inc, Gaithersburg, MD 20878 USA
关键词
D O I
10.2144/04364PF01
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor(TM) nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 72 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA, (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as I in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end-labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.
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页码:702 / +
页数:5
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