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sara_sara新蟲(chóng) (初入文壇)
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[交流]
請(qǐng)教高手基因克隆簡(jiǎn)并引物設(shè)計(jì)的問(wèn)題
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最近想克隆一個(gè)基因,在NCBI上找到了其他幾個(gè)物種中這個(gè)基因的序列,本來(lái)是想設(shè)計(jì)簡(jiǎn)并引物,得到片段,然后再用RACE的方法克隆。可是實(shí)際操作發(fā)現(xiàn)好困難。。 這個(gè)基因A的氨基酸序列C端比較保守,N端差異比較大。因此我想的是從C端找保守區(qū)域設(shè)計(jì)簡(jiǎn)并引物?蓡(wèn)題是,基因A是另一個(gè)基因B的變種(是這么說(shuō)么),這兩個(gè)基因的C端是相似的,區(qū)別就在N端。如果我用C端設(shè)計(jì)引物的話(huà),很可能得到基因B。實(shí)際上我第一次設(shè)計(jì)的引物得到的就是基因B。。。而且這兩個(gè)基因也比較短,就700,800的大小,我不知道應(yīng)該怎么設(shè)計(jì)引物了。。請(qǐng)高手幫忙 ![]() 下面是其他物種中A的氨基酸序列和核苷酸序列 >1 MARTKQMAHKKLRRTLNVDEAGPSTPVTRSTEVNPKSSRPTPVT EDRGTGARKKHRFRPGTVALREIRKYQKTTELLIPAAPFIRLVREITNLYSKEVTRWT PEALLAIQEAAEFFIINLLEEANLCAIHAKRVTLMQKDIQLARRIGGARHFS >2 MARTKHMVKRTIRRKLNVDEAGPSTPATRSAEVKRKKSRPTPVT EERGTGVKKKHRFKAGTVALREIRKYQKTAELLIPAAPFIRLVREITMLYSKEVNRWT PEALLAIQEAAEFFLINLLQEANLCAIHAKRVTVMQKDIQLARRIGGSRHFS >3 MARTKHRVTRSQPRNQTDAAGASSSQAAGPTTTPTRRGGEGGDNTQQTNPTTSPATGTRRGAKRSRQAMPRGSQKKSYRYRPGTVALKEIRHFQKQTNLLIPAASFIREVRSITHMLAPPQINRWTAEALVALQEAAEDYLVGLFSDSMLCAIHARRVTLMRKDFELARRLGGKGRPW >4 MARTKKTVAATKRRSPRTRLEPQSQPEKKKRAHRFRPGTVALRE IRKYRKSTELLIPFAPFVRLVRGIANGFMNIGGPNKPTPWTPHALLSLQEAAEYHLVD LFGKANLCAIHAKRVTVLLKDMRLAKRIGSVTVY >5 MARTKHFSNKKSVRPKKQISGARASSSQVTESTPAKTDAAATRM DSTPASRSIKRTSARKSVAPPQTPTNRGETPQTKERKKHRYRPGTLALRQIRRLQKTT DLLVARAPFARLVREITGHVSKDVNRWQAEALVALQEAAEYYVVNLMEDANLLAIHAR RVTIMQKDIQLARRIGA >6 MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPERAAGTGGRAASGGDSVKKTKPRHRWRPGTVALREIRKYQKSTEPLIPFAPFVRVVRELTNFVTNGKVERYTAEALLALQEAAEFHLIELFEMANLCAIHAKRVTIMQKDIQLARRIGGRRWA 核苷酸序列 >1 GACGATTCAGAGGTCAATCGCAGACGGTTTTTAAATGAAAACGTGAAAACAAACCTTCAAAATGGAGGGATTTGGGATTATTTCAAAATTATCAGTTGTCCCAACGGCAGAATCTTACTCCTCCGAAAATAAAAAATTATAGAATAGAATCAAAATCCTTCTTTGCCCCACTTTCTCCTGGAAAAAAATGGCGAGAACTAAGCAGATGGCTCATAAGAAGCTACGCCGCACGTTGAACGTCGACGAAGCTGGTCCTTCAACACCAGTAACTCGATCGACCGAAGTAAATCCAAAAAGTTCGAGACCTACTCCCGTTACTGAGGATAGAGGGACTGGAGCTCGAAAGAAGCATAGATTCAGGCCAGGCACCGTGGCTCTCCGAGAAATCAGAAAGTACCAAAAAACCACTGAATTACTGAT TCCTGCTGCTCCATTTATCAGGCTTGTGAGGGAGATTACAAATTTGTATTCCAAGGAAGTTACTAGATGGACTCCTGAGGCATTACTTGCCATACAAGAGGCAGCTGAATTTTTTATAATTAACTTGTTGGAAGAGGCAAACTTGTGTGCCATACATGCAAAACGCGTTACACTGATGCAAAAGGATATACAGCTGGCGAGGAGGATTGGTGGTGCTAGACATTTTTCATGATTCAACATCGATGTCATTTACCAGTTTACTAGTAGCTTAGAATATCAATTTGCTCACTAGATTATACTGCTCACTTTAACTAGATTATACTGTTCACTTTGACCATTTGATTATGTTGCCACTTTAACCATTTGATTATGTTGCTCTCTCACTGTTTGTTTACCCATATTATTCAGGTGTATTTTTAG CAGTAGGATTGTATTGTCTGGTAGTTTTATATACTAGAATAATAGTGTGATGGTGCATTTACATTAAAAAAAAAAAAAA >2 AAATTCGAATCAATATCCTTCCTTGCACCCACTTTCTCCTTGAAAAAAAATGGCGAGAACTAAACATATGGTTAAGAGAACGATACGCCGCAAGTTGAACGTCGATGAAGCTGGTCCTTCAACACCAGCAACTCGATCGGCCGAAGTAAAACGGAAAAAATCCAGACCTACTCCCGTCACAGAGGAAAGAGGGACCGGAGTTAAAAAGAAGCATAGATTCAAGGCAGGCACTGTGGCCCTACGGGAAATCAGAAAGTACCAAAAAACTGCAGAATTGCTTATCCCTGCTGCTCCGTTTATCAGGCTTGTGAGGGAAATAACAATGTTGTATTCCAAGGAGGTTAATAGATGGACTCCTGAGGCATTGCTAGCCATACAAGAGGCAGCTGAGTTTTTTCTAATTAACTTGTTGCAAGAGGC AAACTTGTGTGCCATACATGCCAAACGCGTTACAGTTATGCAAAAGGACATTCAGCTGGCGAGGAGGATCGGTGGCTCACGACATTTTTCGTGATTCGATATCGGCATTAGTTACTAATTAACTTGTGTTTTGCTTCTACCTAATTCTTTAACTAGCTGAAACTCTCACTGTTTGTTTACATATTATTCTGGTGTATTCACGTCTAATCAGCATTTTAAGCAATGTTATTAGCTTTATTGTTTGTTTTATACTAGAGTAATAGTGTGATGGTCCTTTTTT >3 AAATTGTGAAGCAGATTCGATTTTAAAATCAAAATTTGAGTGTCGAGCGGGAAAGTAAAAGTTCCGCTCCAATCTTCTAATCTTTTCGTATCTAGCGGGAAATTTCTCAGCAGTAAAAATCAATGGCGAGAACCAAGCATCGCGTTACCAGGTCACAACCTCGGAATCAAACTGATGCCGCCGGTGCTTCATCTTCTCAGGCGGCAGGTCCAACTACGACCCCGACAAGGAGAGGCGGTGAAGGTGGAGATAATACTCAACAAACAAATCCTACAACTTCACCAGCTACTGGTACAAGGAGAGGGGCTAAGAGATCCAGACAGGCTATGCCACGAGGCTCACAGAAGAAGTCTTATCGATACAGGCCAGGAACCGTTGCTCTAAAAGAGATTCGCCATTTCCAGAAGCAGACAAACCTTCTTATTCCGGCTGCCAGTTTCATAAGAGAAGTGAGAAGTATAACCCATATGTTGGCCCCTCCCCAAATCAATCGTTGGACAGCTGAAGCTCTTGTTGCTCTTCAAGAGGCGGCAGAAGATTACTTGGTTGGTTTGTTCTCAGATTCAATGCTCTGTGCTATCCATGCAAGACGTGTTACTCTAATGAGAAAAGACTTTGAACTTGCACGCCGGCTTGGAGGAAAAGGCAGACCATGGTGATAGAAAACTCACTCACTATTCACATCTCTTACATTGTAAGTGAGGATCGAATAGTTAACAACAAGTGTGTCGTAGGTTGTATAAGGTTATTCTCTCCTTGTCTCTTGGTGATGCATGTCCTTTTATTTTACTAAATGATCTGACCAAAACCTTCTTTTGAAACATAACTGGAAACCCGAAT TTGGTTTGATTCCAATATCTAGGCTGTAAAACTTCTTTCTCTATGTGTGTTTCTATCTGCTTTTCGTTG >4 ATGGCTCGCACGAAGAAAACGGTGGCGGCGACCAAGAGGCGCTCCCCTCGCACGAGGTTGGAGCCGCAGTCGCAGCCGGAGAAGAAGAAGCGGGCGCACCGGTTCCGGCCGGGCACGGTGGCGCTGCGGGAGATCCGGAAGTACCGCAAGTCCACCGAACTGCTCATCCCCTTTGCGCCCTTCGTCCGCCTGGTTAGGGGAATCGCCAACGGCTTCATGAATATCGGGGGGCCCAACAAGCCGACGCCATGGACTCCTCACGCGCTCCTCTCGTTGCAAGAGGCTGCAGAGTATCACTTGGTCGATTTATTTGGAAAGGCAAATCTGTGTGCCATTCATGCGAAGCGTGT TACCGTCCTGCTAAAGGACATGCGGCTCGCGAAGCGTATCGGGTCGGTAACGGTTTACTAATGGAGGATGTTTTAGGCATCATAGGGAGCAAACACTGGTGATGCTAATGGTAGATGTTTTCTGTATGAGTGCAATGCGAAGGCGCTGATGGTAGTGATCCAGTATTTCTGTGAGTGTATCATTAGGAAGTAATGTAGGTGTGTTTTCATCCAGGTAGCATGTCTTCGAAGTTGATCCGGTTCGTTGCATGGTTGGGATCATCTGACTTCTCGGCCTTTGCTCCATTTGGATTGATCTAATTCAGACAGAGTTTCTGCAAATGATCCAAGGATATGGGTTGTCAAACAAAAAAAAAAA >5 ATGGCTCGGACGAAACACTTCTCCAATAAAAAGTCAGTTCGTCCTAAGAAGCAGATCTCCGGTGCCCGAGCTAGCAGTTCTCAAGTTACGGAAAGCACTCCTGCTAAAACTGATGCTGCTGCTACTCGTATGGATTCTACTCCAGCGAGTAGATCCATCAAGAGAACTTCAGCTCGCAAGAGTGTGGCTCCGCCACAAACTCCTACGAATCGAGGAGAAACGCCACAAACAAAGGAGAGAAAGAAGCACCGGTACCGACCCGGTACGTTGGCGCTGAGACAGATCAGAAGGCTTCAGAAAACCACCGATCTATTGGTGGCCAGAGCCCCTTTTGCCAGATTGGTTAGAGA GATAACAGGTCATGTCTCGAAAGATGTGAATCGCTGGCAAGCCGAAGCTCTCGTGGCCCTTCAAGAGGCCGCAGAGTACTATGTTGTGAATCTTATGGAGGATGCGAATTTATTGGCTATTCATGCGAGAAGGGTTACTATTATGCAAAAGGACATTCAACTTGCTAGGCGAATAGGTGCATAA >6 AATTCGGCACGAGGCCCTCCCGTCCCGAGAGTTCTGAATCGAAACCGTCGGCCACGAGAGCAGTGCGAGGCGCCCACCGCGATGGCTCGAACCAAGCACCAGGCCGTGAGGAAGACGGCGGAGAAGCCCAAGAAGAAGCTCCAGTTCGAGCGCTCAGGTGGTGCGAGTACCTCGGCGACGCCGGAAAGGGCTGCTGGGACCGGGGGAAGAGCGGCGTCTGGAGGTGACTCAGTTAAGAAGACGAAACCACGCCACCGCTGGCGGCCAGGGACTGTAGCGCTGCGGGAGATCAGGAAGTACCAGAAGTCCACTGAACCGCTCATCCCCTTTGCGCCTTTCGTCCGTGTGGT GAGGGAGTTAACCAATTTCGTAACAAACGGGAAAGTAGAGCGCTATACCGCAGAAGCCCTCCTTGCGCTGCAAGAGGCAGCAGAATTCCACTTGATAGAACTGTTTGAAATGGCGAATCTGTGTGCCATCCATGCCAAGCGTGTCACAATCATGCAAAAGGACATACAACTTGCAAGGCGTATCGGAGGAAGGCGTTGGGCATGATATATAATATCCATTCTGATTGCATCATTCTTGTGAATTTGTTTGTAGGAGCTAGACATTAGTGTTGTTGAATGCTGCATGGTTCCTAATCCTTTTCGCAGTCTAACATCTGTGGAGTTAGTATGTTACATGGCAACAGCTGAACATCTGTGGACTATAACTATATGGCAACAGCCGAAGATTGTGTCTGTGGGATAACTGGTTGTTTTGGTTGC TCTTCAGTAGTTTGTTTGCTTCAGGTAACCATGCTGCGAACTATGATGTTTTCATTCTCGGTTTGCTTCAGCTAACCGAGATCGATTCAGTCTGCAGTATATGGACTATGGAGTAAACTGCATGCTGAAACCCGAACCACTGCTGAAACGGCAGTTGCCAGGATAGCAGGAGGGCCCTTTATGCACAGTGGAATTGAGTAGAGAACTGAGTAAACCATGGTTCTTTCTCCTTTTGAACTGGAACACAAACACAGTTGGATCTTGTTTCTCTTCTTAGGCCATTGTCATCGTGTTTCTTAGGGGTGTAAATGGTATCTGTCCGTATTCGAATTTGATCTATCTAACAAGGC TGAAATCCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 下面是基因B的,這個(gè)基因在各個(gè)物種中都很保守,基本上是一樣的 氨基酸的 1 martkqtark stggkaprkq latkaarksa patggvkkph rfrpgtvalr eirkyqkste 61 llirklpfqr lvreiaqdfk tdlrfqssav aalqeaaeay lvglfedtnl caihakrvti 121 mpkdiqlarr irgera 核苷酸的 1 cagatctcaa ctttctctca aaaatcaaac agtttctatg gctcgtacca agcagacagc 61 taggaaatca accggaggta aagctccaag gaaacaattg gcgacaaaag cggcgaggaa 121 atctgctcca gccaccggag gagtgaagaa acctcacaga ttcagacccg gaaccgtagc 181 tctaagagaa atcaggaagt accagaagag cactgagctt ctgatccgta aacttccttt 241 ccagaggctt gtgagagaga tcgctcagga cttcaagacc gatctccgtt tccagagcag 301 tgccgtcgcc gccctccagg aagctgctga agcttacctc gtcggactct tcgaagacac 361 caatctctgc gctattcatg ccaagagagt caccatcatg cccaaggaca tccagctcgc 421 aagaagaatc agaggagaaa gagcttagat ctaagtattc tggt |
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