Polymorphic microsatellite loci in Chinese piebald odorous frog (Odorrana schmackeri)
Polymorphic microsatellite loci in Chinese piebald odorous frog (Odorrana schmackeri)
复制标题
中国花斑臭蛙（Odorrana schmackeri）的多态性微卫星位点
DOI：
10.1007/s12041-011-0056-z
复制
发表时间：
2013-12
影响因子：
1.5
通讯作者：
Ding Ping
中科院分区：
文献类型：
--
作者：
Wang Yu;Ding Ping
材料与方法从中国杭州采集臭蛙趾样品30个。使用标准蛋白酶K和苯酚-氯仿方案分离基因组DNA（Sambrook等人1989; Wang等人2009）。使用Shao等人的方案富集微卫星DNA片段。（2009年）。用限制酶Sau 3AI（Takara，Dalian，PR China）消化基因组DNA，然后使用DNA凝胶提取试剂盒（Axygen，Hangzhou，PR China）分离和纯化300-1000-bp片段。使用T4 DNA连接酶（Takara，Dalian，PR China）将双链Sau 3A衔接子（Sau 3AL：5-GGC CAG AGA CCC CAA GCT TCG-3和Sau 3AR：5-GAT CCG AAG CTT GGG GTC TCT GGC C-3）连接到DNA片段，然后通过聚合酶链式反应（PCR）用特异性引物Sau 3AL扩增接头连接的片段以测试连接效率。将具有接头的片段与生物素标记的二核苷酸重复序列（CA）15探针杂交。最后，通过磁性链霉亲和素珠（Promega，Beijing，PR China）捕获微卫星富集的DNA片段。将纯化的PCR产物（富含微卫星的DNA片段）连接到pMD 19-T载体（Takara，Dalian，PR China）中，然后转化到E. coli DH 5 α感受态细胞（Takara，Dalian，PR China）。转化的细胞在LB/IPTG +/X-Gal平板上于37 ℃生长16 h，进行蓝/白色选择以鉴定阳性克隆。用引物Sau 3AL和寡核苷酸（CA）15扩增总共576个阳性克隆，然后使用3730 Bigdye-终止子（ABI，Foster City，USA）测序。使用引物5（Lalitha 2000）设计引物。总共测试了45对引物进行扩增，其中8对扩增出一致的产物。所有样本均用于检测多态性。反应体积，10 μL，
Materials and methodsIn total, 30 toe samples of Odorrana schmackeri were collected from Hangzhou, PR China. Genomic DNA was isolated using a standard proteinase K and phenol–chloroform protocol (Sambrook et al. 1989; Wang et al. 2009). Microsatellite DNA fragments were enriched using the protocol of Shao et al.(2009). Genomic DNA was digested with the restriction enzyme Sau3AI (Takara, Dalian, PR China), then 300–1000-bp fragments were isolated and purified using a DNA Gel Extraction kit (Axygen, Hangzhou, PR China). Double-stranded Sau3A adapters (Sau3AL: 5-GGC CAG AGA CCC CAA GCT TCG-3 and Sau3AR: 5-GAT CCG AAG CTT GGG GTC TCT GGC C-3) were ligated to DNA fragments using the T4 DNA ligase (Takara, Dalian, PR China), and the linker-ligated fragments were then amplified with specific primer Sau3AL by polymerase chain reaction (PCR) to test the ligation efficiency. The fragments with adapters were hybridized with biotin-labelled dinucleotide repeat sequences (CA) 15 probes. Finally, the microsatellite-enriched DNA fragments were captured by magnetic streptavidin beads (Promega, Beijing, PR China). Purified PCR products (microsatellite-enriched DNA fragments) were ligated into the pMD19-T vector (Takara, Dalian, PR China) and then transformed into E. coli DH5α competent cells (Takara, Dalian, PR China). Transformed cells grew at 37◦ C for 16 h on LB/Amp+/IPTG/X-Gal plates for blue/white selection to identify positive clones. In total, 576 positive clones were amplified with the primer Sau3AL and oligonucleotides (CA) 15, then sequenced using the 3730 Bigdye-Terminator (ABI, Foster City, USA). Primers were designed using Primer 5 (Lalitha 2000). In total, 45 primer pairs were tested for amplification, eight of which amplified a consistent product. All samples were used to test for polymorphisms. Reaction volume, 10-μL,
登录
查看更多内容
影响因子：
4.9
作者：
T. Knopp;J. Merilä
通讯作者：
T. Knopp;J. Merilä
影响因子：
2.2
作者：
Chen Shao;Yu Wang;N. Qiao
通讯作者：
Chen Shao;Yu Wang;N. Qiao
影响因子：
4.9
作者：
M. Murphy;R. Dezzani;D. Pilliod;Andrew Storfer
通讯作者：
M. Murphy;R. Dezzani;D. Pilliod;Andrew Storfer
DOI：
--
发表时间：
2001-01
期刊：
--
影响因子：
--
作者：
J. Sambrook;E. Fritsch;T. Maniatis
通讯作者：
J. Sambrook;E. Fritsch;T. Maniatis
DOI：
10.1111/j.1471-8286.2004.00684.x
发表时间：
2004-09-01
期刊：
MOLECULAR ECOLOGY NOTES
影响因子：
--
作者：
Van Oosterhout, C;Hutchinson, WF;Shipley, P
通讯作者：
Shipley, P