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        各位大侠,帮我设计一个引物

        丁香园论坛

        521
        我作RT-PCR, 这个引物实在是设计不好,是TOPO II ALPHA
        其mRNA如下:1 aggttcaagt ggagctctcc taaccgacgc gcgtctgtgg agaagcggct tggtcggggg
        61 tggtctcgtg gggtcctgcc tgtttagtcg ctttcagggt tcttgagccc cttcacgacc
        121 gtcaccatgg aagtgtcacc attgcagcct gtaaatgaaa atatgcaagt caacaaaata
        181 aagaaaaatg aagatgctaa gaaaagactg tctgttgaaa gaatctatca aaagaaaaca
        241 caattggaac atattttgct ccgcccagac acctacattg gttctgtgga attagtgacc
        301 cagcaaatgt gggtttacga tgaagatgtt ggcattaact atagggaagt cacttttgtt
        361 cctggtttgt acaaaatctt tgatgagatt ctagttaatg ctgcggacaa caaacaaagg
        421 gacccaaaaa tgtcttgtat tagagtcaca attgatccgg aaaacaattt aattagtata
        481 tggaataatg gaaaaggtat tcctgttgtt gaacacaaag ttgaaaagat gtatgtccca
        541 gctctcatat ttggacagct cctaacttct agtaactatg atgatgatga aaagaaagtg
        601 acaggtggtc gaaatggcta tggagccaaa ttgtgtaaca tattcagtac caaatttact
        661 gtggaaacag ccagtagaga atacaagaaa atgttcaaac agacatggat ggataatatg
        721 ggaagagctg gtgagatgga actcaagccc ttcaatggag aagattatac atgtatcacc
        781 tttcagcctg atttgtctaa gtttaaaatg caaagcctgg acaaagatat tgttgcacta
        841 atggtcagaa gagcatatga tattgctgga tccaccaaag atgtcaaagt ctttcttaat
        901 ggaaataaac tgccagtaaa aggatttcgt agttatgtgg acatgtattt gaaggacaag
        961 ttggatgaaa ctggtaactc cttgaaagta atacatgaac aagtaaacca caggtgggaa
        1021 gtgtgtttaa ctatgagtga aaaaggcttt cagcaaatta gctttgtcaa cagcattgct
        1081 acatccaagg gtggcagaca tgttgattat gtagctgatc agattgtgac taaacttgtt
        1141 gatgttgtga agaagaagaa caagggtggt gttgcagtaa aagcacatca ggtgaaaaat
        1201 cacatgtgga tttttgtaaa tgccttaatt gaaaacccaa cctttgactc tcagacaaaa
        1261 gaaaacatga ctttacaacc caagagcttt ggatcaacat gccaattgag tgaaaaattt
        1321 atcaaagctg ccattggctg tggtattgta gaaagcatac taaactgggt gaagtttaag
        1381 gcccaagtcc agttaaacaa gaagtgttca gctgtaaaac ataatagaat caagggaatt
        1441 cccaaactcg atgatgccaa tgatgcaggg ggccgaaact ccactgagtg tacgcttatc
        1501 ctgactgagg gagattcagc caaaactttg gctgtttcag gccttggtgt ggttgggaga
        1561 gacaaatatg gggttttccc tcttagagga aaaatactca atgttcgaga agcttctcat
        1621 aagcagatca tggaaaatgc tgagattaac aatatcatca agattgtggg tcttcagtac
        1681 aagaaaaact atgaagatga agattcattg aagacgcttc gttatgggaa gataatgatt
        1741 atgacagatc aggaccaaga tggttcccac atcaaaggct tgctgattaa ttttatccat
        1801 cacaactggc cctctcttct gcgacatcgt tttctggagg aatttatcac tcccattgta
        1861 aaggtatcta aaaacaagca agaaatggca ttttacagcc ttcctgaatt tgaagagtgg
        1921 aagagttcta ctccaaatca taaaaaatgg aaagtcaaat attacaaagg tttgggcacc
        1981 agcacatcaa aggaagctaa agaatacttt gcagatatga aaagacatcg tatccagttc
        2041 aaatattctg gtcctgaaga tgatgctgct atcagcctgg cctttagcaa aaaacagata
        2101 gatgatcgaa aggaatggtt aactaatttc atggaggata gaagacaacg aaagttactt
        2161 gggcttcctg aggattactt gtatggacaa actaccacat atctgacata taatgacttc
        2221 atcaacaagg aacttatctt gttctcaaat tctgataacg agagatctat cccttctatg
        2281 gtggatggtt tgaaaccagg tcagagaaag gttttgttta cttgcttcaa acggaatgac
        2341 aagcgagaag taaaggttgc ccaattagct ggatcagtgg ctgaaatgtc ttcttatcat
        2401 catggtgaga tgtcactaat gatgaccatt atcaatttgg ctcagaattt tgtgggtagc
        2461 aataatctaa acctcttgca gcccattggt cagtttggta ccaggctaca tggtggcaag
        2521 gattctgcta gtccacgata catctttaca atgctcagct ctttggctcg attgttattt
        2581 ccaccaaaag atgatcacac gttgaagttt ttatatgatg acaaccagcg tgttgagcct
        2641 gaatggtaca ttcctattat tcccatggtg ctgataaatg gtgctgaagg aatcggtact
        2701 gggtggtcct gcaaaatccc caactttgat gtgcgtgaaa ttgtaaataa catcaggcgt
        2761 ttgatggatg gagaagaacc tttgccaatg cttccaagtt acaagaactt caagggtact
        2821 attgaagaac tggctccaaa tcaatatgtg attagtggtg aagtagctat tcttaattct
        2881 acaaccattg aaatctcaga gcttcccgtc agaacatgga cccagacata caaagaacaa
        2941 gttctagaac ccatgttgaa tggcaccgag aagacacctc ctctcataac agactatagg
        3001 gaataccata cagataccac tgtgaaattt gttgtgaaga tgactgaaga aaaactggca
        3061 gaggcagaga gagttggact acacaaagtc ttcaaactcc aaactagtct cacatgcaac
        3121 tctatggtgc tttttgacca cgtaggctgt ttaaagaaat atgacacggt gttggatatt
        3181 ctaagagact tttttgaact cagacttaaa tattatggat taagaaaaga atggctccta
        3241 ggaatgcttg gtgctgaatc tgctaaactg aataatcagg ctcgctttat cttagagaaa
        3301 atagatggca aaataatcat tgaaaataag cctaagaaag aattaattaa agttctgatt
        3361 cagaggggat atgattcgga tcctgtgaag gcctggaaag aagcccagca aaaggttcca
        3421 gatgaagaag aaaatgaaga gagtgacaac gaaaaggaaa ctgaaaagag tgactccgta
        3481 acagattctg gaccaacctt caactatctt cttgatatgc ccctttggta tttaaccaag
        3541 gaaaagaaag atgaactctg caggctaaga aatgaaaaag aacaagagct ggacacatta
        3601 aaaagaaaga gtccatcaga tttgtggaaa gaagacttgg ctacatttat tgaagaattg
        3661 gaggctgttg aagccaagga aaaacaagat gaacaagtcg gacttcctgg gaaagggggg
        3721 aaggccaagg ggaaaaaaac acaaatggct gaagttttgc cttctccgcg tggtcaaaga
        3781 gtcattccac gaataaccat agaaatgaaa gcagaggcag aaaagaaaaa taaaaagaaa
        3841 attaagaatg aaaatactga aggaagccct caagaagatg gtgtggaact agaaggccta
        3901 aaacaaagat tagaaaagaa acagaaaaga gaaccaggta caaagacaaa gaaacaaact
        3961 acattggcat ttaagccaat caaaaaagga aagaagagaa atccctggtc tgattcagaa
        4021 tcagatagga gcagtgacga aagtaatttt gatgtccctc cacgagaaac agagccacgg
        4081 agagcagcaa caaaaacaaa attcacaatg gatttggatt cagatgaaga tttctcagat
        4141 tttgatgaaa aaactgatga tgaagatttt gtcccatcag atgctagtcc acctaagacc
        4201 aaaacttccc caaaacttag taacaaagaa ctgaaaccac agaaaagtgt cgtgtcagac
        4261 cttgaagctg atgatgttaa gggcagtgta ccactgtctt caagccctcc tgctacacat
        4321 ttcccagatg aaactgaaat tacaaaccca gttcctaaaa agaatgtgac agtgaagaag
        4381 acagcagcaa aaagtcagtc ttccacctcc actaccggtg ccaaaaaaag ggctgcccca
        4441 aaaggaacta aaagggatcc agctttgaat tctggtgtct ctcaaaagcc tgatcctgcc
        4501 aaaaccaaga atcgccgcaa aaggaagcca tccacttctg atgattctga ctctaatttt
        4561 gagaaaattg tttcgaaagc agtcacaagc aagaaatcca agggggagag tgatgacttc
        4621 catatggact ttgactcagc tgtggctcct cgggcaaaat ctgtacgggc aaagaaacct
        4681 ataaagtacc tggaagagtc agatgaagat gatctgtttt aaaatgtgag gcgattattt
        4741 taagtaatta tcttaccaag cccaagactg gttttaaagt tacctgaagc tcttaacttc
        4801 ctcccctctg aatttagttt ggggaaggtg tttttagtac aagacatcaa agtgaagtaa
        4861 agcccaagtg ttctttagct ttttataata ctgtctaaat agtgaccatc tcatgggcat
        4921 tgttttcttc tctgctttgt ctgtgttttg agtctgcttt cttttgtctt taaaacctga
        4981 tttttaagtt cttctgaact gtagaaatag ctatctgatc acttcagcgt aaagcagtgt
        5041 gtttattaac catccactaa gctaaaacta gagcagtttg atttaaaagt gtcactcttc
        5101 ctccttttct actttcagta gatatgagat agagcataat tatctgtttt atcttagttt
        5161 tatacataat ttaccatcag atagaacttt atggttctag tacagatact ctactacact
        5221 cagcctctta tgtgccaagt ttttctttaa gcaatgagaa attgctcatg ttcttcatct
        5281 tctcaaatca tcagaggcca aagaaaaaca ctttggctgt gtctataact tgacacagtc
        5341 aatagaatga agaaaattag agtagttatg tgattatttc agctcttgac ctgtcccctc
        5401 tggctgcctc tgagtctgaa tctcccaaag agagaaacca atttctaaga ggactggatt
        5461 gcagaagact cggggacaac atttgatcca agatcttaaa tgttatattg ataaccatgc
        5521 tcagcaatga gctattagat tcattttggg aaatctccat aatttcaatt tgtaaacttt
        5581 gttaagacct gtctacattg ttatatgtgt gtgacttgag taatgttatc aacgtttttg
        5641 taaatattta ctatgttttt ctattagcta aattccaaca attttgtact ttaataaa
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