各位大侠，帮我设计一个引物
丁香园论坛
409
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
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
