摘要
用2种方法克隆黑龙江鲤(Cyprinus(Cyprinus)carpiohaematopterus)的微卫星序列。这2种方法分别是:①经典的小片段DNA克隆库,用末端标记的[γ-32]ATP的CA重复序列为探针筛选;②将酶切获得的小片段DNA用结合有磁珠的并连接有15个CA重复序列的生物素进行富集,获得的含有CA重复的DNA片段并经过两次PCR扩增再克隆的方法获得富集微卫星片段的克隆库。从前一个方法的克隆库中筛选2000个菌落,获得阳性克隆45个,有22个含有微卫星,完美型的占63 6%,非完美型的占22 7%,混合型的占13 7%,重复次数超过10的有9个,占40 9%;从方法②的克隆库中筛选2600个菌落,获得阳性克隆1300个,测序其中的390个克隆,微卫星314个,完美型的占79 0%;非完美型的占14 3%;混合型的占6 7%,重复次数超过10的有293个,占93 3%。结果表明,用生物素结合磁珠富集法克隆微卫星效率高,成本低,所获微卫星质量高,是一种值得推荐的微卫星制备方法。
Microsatellites are important markers for development of genetic maps,germ plasma assessment,quantitative trait loci mapping because of their high polymorphism,abundance,co-dominance,and small locus size.In aquatic species,microsatellite markers have been used for salmon,tilapia,rainbow trout,catfish etc.on genetic map,quantitative trait loci mapping,and population genetics assessment.But only a few local aquaculture species in China,their microsatellite markers have been developed.Because up till now there are not enough micrksatellite markers,the technique of RAPD marker is still used in genetic assessment for some important aquatic species such as river crab,common carp,silver crucian carp,silver carp etc.For genetic assessments research,the marker that identifies different populations in one species is very important.Owing to its co-dominant property,microsatellite marker should be a good candidate for this kind of marker.In this test,some microsatellite sequences for common carp were cloned by two methods.One was that traditional inserted DNA fragments libraries were screened by plaque hybridization using oligos (CA)_15 as probes,end-labeled with [γ-32] ATP.The other was that inserted DNA fragments were linked onto linkers and then enriched microsatellite sequences with magnetic beads which was linked with biotinylated (CA)_15 probes,finally the PCR-based library was made.2000 colones from the former method were selected and screened,and 45 colones were positive.In those positive colones,22 microsatellite sequences were gotten.Among them,perfect was 63.6%,imperfect was 22.7%,compound was 13.7%,repeated numbers over 10 were 9 and about 40.9%.About 2600 colones from the latter method were selected and screened,and 1300 colones were positive.From those positive colones,390 colones were sequenced,and 314 microsatellite sequences were gotten.In those microsatellites,perfect was 79.0%,imperfect was 14.3%,compound was 6.7%,and repeated numbers over 10 were 293 and about 93.3%.This result showed that the magnetic beads enriched method could be a high efficiency and low cost method,and microsatellite sequences from it could be of high quality.So,we recommended that magnetic beads enriched method be used in making microsatellite markers.
出处
《中国水产科学》
CAS
CSCD
北大核心
2005年第2期126-132,共7页
Journal of Fishery Sciences of China
基金
国家"863"高技术研究发展项目(863-101-05-02-01).
关键词
磁珠富集
小片段克隆
鲤
微卫星
magnetic beads enrichment
small fragments DNA cloning
common carp
microsatellite
