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标题: 已知一条cDNA序列，能否反推其内含子插入的位置 [打印本页]

作者: loli 时间: 2011-9-16 17:39 标题: 已知一条cDNA序列，能否反推其内含子插入的位置

已知一条cDNA序列，能否反推其内含子插入的位置 [转载]

问题的背景：我得到了一条cDNA序列，想设计一对引物进行RT—PCR mRNA半定量，为了预防genomic DNA的污染，两引物中间最好不能有intron，或者说两引物要跨越不同的exon上。

问题：我如何能知道我所得到cDNA序列中内含子插入的位置？是否通过软件可以找得到？如果可以的话，DNAstar软件能否可以完成该功能？

请各位大侠指教。

谢谢！

作者: 不懂 时间: 2011-9-16 17:39

just check GenBank.
the genomics of human,rat and mouse are all very clear.

作者: 不懂 时间: 2011-9-16 17:40

http://genome.ucsc.edu/,比NCBI要好查。先选你要查的基因组，直接输入你的cDNA序列进行Blat search即可

作者: 丁香@@ 时间: 2011-9-16 17:41

just check GenBank.
the genomics of human,rat and mouse are all very clear.

=======================================================

哪位高手详细讲一下？毕竟熟悉genbank的就不问这样的问题了。谢谢！

作者: fangxiang 时间: 2011-9-16 17:41

问题的背景：我得到了一条cDNA序列，想设计一对引物进行RT—PCR mRNA半定量，为了预防genomic DNA的污染，两引物中间最好不能有intron，或者说两引物要跨越不同的exon上。

问题：我如何能知道我所得到cDNA序列中内含子插入的位置？是否通过软件可以找得到？如果可以的话，DNAstar软件能否可以完成该功能？

请各位大侠指教。

谢谢！

作者: fangxiang 时间: 2011-9-16 17:42

dnastar 完全可以完成这个任务，请使用genequest模块，显示结果如下：

图片附件: 31883158.png (2011-9-16 17:42, 16.37 KB) / 该附件被下载次数 7
http://bbs.antpedia.com/attachment.php?aid=8584

作者: fangxiang 时间: 2011-9-16 17:43

下面是blast后的结果，所有能够比对成功的都是外显子序列，每一个比对组都是一个外显子，引物就在里面找就行了。

Alignments

>ref|NT_039655.2|Mm17_39695_32 Mus musculus chromosome 17 genomic contig, strain C57BL/6J
Length = 13907576

Score = 681 bits (354), Expect = 0.0
Identities = 354/354 (100%)
Strand = Plus / Plus

Query: 202 cagccctgtcccaagccctcaacaccacggtgctgcagggcatggccggccagtccttga 261
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8131902 cagccctgtcccaagccctcaacaccacggtgctgcagggcatggccggccagtccttga 8131961

Query: 262 gggtgtcatgtacttatgacgccttgaagcactgggggagacgcaaggcctggtgtcggc 321
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8131962 gggtgtcatgtacttatgacgccttgaagcactgggggagacgcaaggcctggtgtcggc 8132021

Query: 322 agctgggtgaggagggcccatgccagcgtgtggtgagcacacacggtgtgtggctgctgg 381
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8132022 agctgggtgaggagggcccatgccagcgtgtggtgagcacacacggtgtgtggctgctgg 8132081

Query: 382 ccttcctgaagaagcggaatgggagcacagtcatcgcagatgacacccttgctggaaccg 441
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8132082 ccttcctgaagaagcggaatgggagcacagtcatcgcagatgacacccttgctggaaccg 8132141

作者: fangxiang 时间: 2011-9-16 17:43

Query: 442 tcaccatcactctgaagaacctccaagccggtgacgcgggcctctaccagtgtcagagtc 501
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8132142 tcaccatcactctgaagaacctccaagccggtgacgcgggcctctaccagtgtcagagtc 8132201

Query: 502 tccgaggccgagaggctgaggtcctgcagaaagtactggtggaggtgctggagg 555
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8132202 tccgaggccgagaggctgaggtcctgcagaaagtactggtggaggtgctggagg 8132255

Score = 514 bits (267), Expect = e-143
Identities = 267/267 (100%)
Strand = Plus / Plus

Query: 831 ggacccggaggtacgtgagagaattctgagtgggaggagaactacagcttaagtccagcc 890
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135455 ggacccggaggtacgtgagagaattctgagtgggaggagaactacagcttaagtccagcc 8135514

Query: 891 aggagtcaatccagcctgcatgctctcccctcctccaccaagacttctgtttctgctact 950
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135515 aggagtcaatccagcctgcatgctctcccctcctccaccaagacttctgtttctgctact 8135574

Query: 951 tttgcttcagaggccgcctctgcctcaagcccacctatcctgggagcaggaatactggtg 1010
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135575 tttgcttcagaggccgcctctgcctcaagcccacctatcctgggagcaggaatactggtg 8135634

Query: 1011 tgtacatctgtgttgagtggggaagacagctggatggttgtctgtcaacttctgcacttt 1070
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135635 tgtacatctgtgttgagtggggaagacagctggatggttgtctgtcaacttctgcacttt 8135694

Query: 1071 ggacattaaacattctccacacaccaa 1097
|||||||||||||||||||||||||||
Sbjct: 8135695 ggacattaaacattctccacacaccaa 8135721

Score = 375 bits (195), Expect = e-101
Identities = 195/195 (100%)
Strand = Plus / Plus

Query: 10 gcgcctaccctagtcctgactgttgctcaatccaggagcacagttcctgtgggctgagcc 69
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8129849 gcgcctaccctagtcctgactgttgctcaatccaggagcacagttcctgtgggctgagcc 8129908

作者: fangxiang 时间: 2011-9-16 17:44

Query: 70 tgactggcttggtcatctcttttctgcacttcaagggaaaagcaagatcttgcacaaggt 129
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8129909 tgactggcttggtcatctcttttctgcacttcaagggaaaagcaagatcttgcacaaggt 8129968

Query: 130 cccctccggctggctgctggcaaaggaaaggtgccatgggacctctccaccagtttctcc 189
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8129969 cccctccggctggctgctggcaaaggaaaggtgccatgggacctctccaccagtttctcc 8130028

Query: 190 tgctgctgatcacag 204
|||||||||||||||
Sbjct: 8130029 tgctgctgatcacag 8130043

Score = 362 bits (188), Expect = 3e-97
Identities = 191/192 (99%), Gaps = 1/192 (0%)
Strand = Plus / Plus

Query: 642 tcc-aggaatcaagagacctccttcccacccacctccattcttctcctcctggcctgcgt 700
||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135188 tccaaggaatcaagagacctccttcccacccacctccattcttctcctcctggcctgcgt 8135247

Query: 701 tctcctgagcaagtttcttgcagccagcatcctctgggctgtggccaggggcaggcagaa 760
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135248 tctcctgagcaagtttcttgcagccagcatcctctgggctgtggccaggggcaggcagaa 8135307

Query: 761 gccgggaacacctgtggtcagagggctggactgtggccaagatgctgggcaccaacttca 820
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8135308 gccgggaacacctgtggtcagagggctggactgtggccaagatgctgggcaccaacttca 8135367

Query: 821 gatcctcactgg 832
||||||||||||
Sbjct: 8135368 gatcctcactgg 8135379

Score = 179 bits (93), Expect = 3e-42
Identities = 93/93 (100%)
Strand = Plus / Plus

Query: 555 gaccctctagatgaccaagatgctggagatctctgggtccccgaggagtcatcgagtttc 614
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 8134548 gaccctctagatgaccaagatgctggagatctctgggtccccgaggagtcatcgagtttc 8134607

Query: 615 gagggtgcccaagtggaacacagcacctccagg 647
|||||||||||||||||||||||||||||||||
Sbjct: 8134608 gagggtgcccaagtggaacacagcacctccagg 8134640

作者: fangxiang 时间: 2011-9-16 17:44

Database: contig
Posted date: Oct 31, 2003 2:42 PM
Number of letters in database: -1,473,234,923
Number of sequences in database: 31,506

Lambda K H
1.33 0.621 1.12

Gapped
Lambda K H
1.33 0.621 1.12

Matrix: blastn matrix:1 -2
Gap Penalties: Existence: 0, Extension: 0
Number of Hits to DB: 13,731
Number of Sequences: 31506
Number of extensions: 0
Number of successful extensions: 0
Number of sequences better than 1.0e-02: 0
Number of HSP's better than 0.0 without gapping: 0
Number of HSP's successfully gapped in prelim test: 0
Number of HSP's that attempted gapping in prelim test: 0
Number of HSP's gapped (non-prelim): 0
length of query: 2258
length of database: 2,821,732,369
effective HSP length: 24
effective length of query: 1104
effective length of database: 2,820,976,225
effective search space: 3114357752400
effective search space used: 0
S2: 25 (48.8 bits)

作者: fangxiang 时间: 2011-9-16 17:44

此外，你还可以使用locuslink检索，具体看看下面的贴子：
cuturl('http://www.dxy.cn/bbs/post/view?bid=64&id=596287&tpg=1&ppg=1&sty=1&age=0#596287')
cuturl('http://www.dxy.cn/bbs/post/view?bid=73&id=594588&sty=3&keywords=locuslink')
cuturl('http://www.dxy.cn/bbs/post/view?bid=73&id=594678&tpg=1&ppg=1&sty=1&age=0#594678')

作者: 8黑眼圈8 时间: 2011-9-16 17:45

谢谢imsupergene 的解释，也欢迎你来paltalk语音交流版做客！
cuturl('http://www.dxy.cn/bbs/post/page?bid=109&sty=1&age=0')

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