请哪位引物设计高手帮忙，我急求特异性高的引物。急！急！急！
丁香园论坛
838
基因序列如下：
1 ctcctg gtgcaatgga gcgagtatta aaggtctttc attattttga aagcaatagt
gagccaacca ccgaccactgtg agcccgcggc gtgaggcgtg ggaggaagcg cggctgctgt
cgcccagcgc
61 cgccccgtcg tcgtctgcct tcgcttcacg gcgccgagcc gcggtccgaa gtcttgctgt
121 gtcacccagg ctgccaggct ggagtggagt ggcatgatct cggctgactg caacctctgc
181 ctcccagaat atgacagata cctagcatct agcaaaataa tggcagctgc ttaccttgac
241 cccaacttga atcacacacc aaattcgagt actaagactc acctgggtac tggtatggaa
301 cgtttgggccag tattatcagg catggagatg ctactgatgt caggggcatc
421 attcagaaga tagtggacag tcacaaagta aagcatgtgg cctgctatgg attccgcctc
481 agtcacctgc ggtcagagga ggttcactgg cttcacgtgg atatgggcgt ctccagtgtg
541 agggagaagt atgagcttgc tcacccacca gaggagtgga aatatgaatt gagaattcgt
601 tatttgccaa aaggatttct aaaccagttt actgaagata agccaacttt gaatttcttc
661 tatcaacagg tgaagagcga ttatatgtta gagatagctg atcaagtgga ccaggaaatt
721 gctttgaagt tgggttgtct agaaatacgg cgatcatact gggagatgcg gggcaatgca
781 ctagaaaaga agtctaacta tgaagtatta gaaaaagatg ttggtttaaa gcgatttttt
841 cctaagagtt tactggattc tgtcaaggcc aaaacactaa gaaaactgat ccaacaaaca
901 tttagacaat ttgccaacct taatagagaa gaaagtattc tgaaattctt tgagatcctg
961 tctccagtct acagatttga taaggaatgc ttcaagtgtg ctcttggttc aagctggatt
> 1021 atttcagtgg aactggcaat cggcccagaa gaaggaatca gttacctaac ggacaagggc
> 1081 tgcaatccca cacatcttgc tgacttcact caagtgcaaa ccattcagta ttcaaacagt
> 1141 gaagacaagg acagaaaagg aatgctacaa ctaaaaatag caggtgcacc cgagcctctg
> 1201 acagtgacgg caccatccct aaccattgcg gagaatatgg ctgacctaat agatgggtac
> 1261 tgccggctgg tgaatggaac ctcgcagtca tttatcatca gacctcagaa agaaggtgaa
> 1321 cgggctttgc catcaatacc aaagttggcc aacagcgaaa agcaaggcat gcggacacac
> 1381 gccgtctctg tgtcagaaac agatgattat gctgagatta tagatgaaga agatacttac
> 1441 accatgccct caaccaggga ttatgagatt caaagagaaa gaatagaact tggacgatgt
> 1501 attggagaag gccaatttgg agatgtacat caaggcattt atatgagtcc agagaatcca
> 1561 gctttggcgg ttgcaattaa aacatgtaaa aactgtactt cggacagcgt gagagagaaa
> 1621 tttcttcaag aagccttaac aatgcgtcag tttgaccatc ctcatattgt gaagctgatt
> 1681 ggagtcatca cagagaatcc tgtctggata atcatggagc tgtgcacact tggagagctg
> 1741 aggtcatttt tgcaagtaag gaaatacagt ttggatctag catctttgat cctgtatgcc
> 1801 tatcagctta gtacagctct tgcatatcta gagagcaaaa gatttgtaca cagggacatt
> 1861 gctgctcgga atgttctggt gtcctcaaat gattgtgtaa aattaggaga ctttggatta
> 1921 tcccgatata tggaagatag tacttactac aaagcttcca aaggaaaatt gcctattaaa
> 1981 tggatggctc cagagtcaat caattttcga cgttttacct cagctagtga cgtatggatg
> 2041 tttggtgtgt gtatgtggga gatactgatg catggtgtga agccttttca aggagtgaag
> 2101 aacaatgatg taatcggtcg aattgaaaat ggggaaagat taccaatgcc tccaaattgt
> 2161 cctcctaccc tctacagcct tatgacgaaa tgctgggcct atgaccccag caggcggccc
> 2221 aggtttactg aacttaaagc tcagctcagc acaatcctgg aggaagagaa ggctcagcaa
> 2281 gaagagcgca tgaggatgga gtccagaaga caggccacag tgtcctggga ctccggaggg
> 2341 tctgatgaag caccgcccaa gcccagcaga ccgggttatc ccagtccgag gtccagcgaa
> 2401 ggattttatc ccagcccaca gcacatggta caaaccaatc attaccaggt ttctggctac
> 2461 cctggttcac atggaatcac agccatggct ggcagcatct atccaggtca ggcatctctt
> 2521 ttggaccaaa cagattcatg gaatcataga cctcaggaga tagcaatgtg gcagcccaat
> 2581 gtggaggact ctacagtatt ggacctgcga gggattgggc aagtgttgcc aacccatctg
> 2641 atggaagagc gtctaatccg acagcaacag gaaatggaag aagatcagcg ctggctggaa
> 2701 aaagaggaaa gatttctgaa acctgatgtg agactctctc gaggcagtat tgacagggag
> 2761 gatggaagtc ttcagggtcc gattggaaac caacatatat atcagcctgt gggtaaacca
> 2821 gatcctgcag ctccaccaaa gaaaccgcct cgccctggag ctcccggtca tctgggaagc
> 2881 cttgccagcc tcagcagccc tgctgacagc tacaacgagg gtgtcaagct tcagccccag
> 2941 gaaatcagcc cccctcctac tgccaacctg gaccggtcga atgataaggt gtacgagaat
> 3001 gtgacgggcc tggtgaaagc tgtcatcgag atgtccagta aaatccagcc agccccacca
> 3061 gaggagtatg tccctatggt gaaggaagtc ggcttggccc tgaggacatt attggccact
> 3121 gtggatgaga ccattcccct cctaccagcc agcacccacc gagagattga gatggcacag
> 3181 aagctattga actctgacct gggtgagctc atcaacaaga tgaaactggc ccagcagtat
> 3241 gtcatgacca gcctccagca agagtacaaa aagcaaatgc tgactgctgc tcacgccctg
> 3301 gctgtggatg ccaaaaactt actcgatgtc attgaccaag caagactgaa aatgcttggg
> 3361 cagacgagac cacactgagc ctcccctagg agcacgtctt gctaccctct tttgaagatg
> 3421 ttctctagcc ttccaccagc agcgaggaat taaccctgtg tcctcagtcg ccagcactta
> 3481 cagctccaac ttttttgaat gaccatctgg ttgaaaaatc tttctcatat aagtttaacc
> 3541 acactttgat ttgggttcat tttttgtttt gtttttttca atcatgatat tcagaaaaat
> 其中我要研究的多态性序列为GAATCATAGA CCTCAGGAGA,等位基因为该图第2551位点的C(既C/T),所以需要针对该多态性序列设计一对引物并合成。pcr后的基因片段最好是300-500之间。我已经试过两对了，都没有成功，因为特异性太差。所以我觉得排除同源序列很重要。
e-mail : bangbanglu@163.com
1 ctcctg gtgcaatgga gcgagtatta aaggtctttc attattttga aagcaatagt
gagccaacca ccgaccactgtg agcccgcggc gtgaggcgtg ggaggaagcg cggctgctgt
cgcccagcgc
61 cgccccgtcg tcgtctgcct tcgcttcacg gcgccgagcc gcggtccgaa gtcttgctgt
121 gtcacccagg ctgccaggct ggagtggagt ggcatgatct cggctgactg caacctctgc
181 ctcccagaat atgacagata cctagcatct agcaaaataa tggcagctgc ttaccttgac
241 cccaacttga atcacacacc aaattcgagt actaagactc acctgggtac tggtatggaa
301 cgtttgggccag tattatcagg catggagatg ctactgatgt caggggcatc
421 attcagaaga tagtggacag tcacaaagta aagcatgtgg cctgctatgg attccgcctc
481 agtcacctgc ggtcagagga ggttcactgg cttcacgtgg atatgggcgt ctccagtgtg
541 agggagaagt atgagcttgc tcacccacca gaggagtgga aatatgaatt gagaattcgt
601 tatttgccaa aaggatttct aaaccagttt actgaagata agccaacttt gaatttcttc
661 tatcaacagg tgaagagcga ttatatgtta gagatagctg atcaagtgga ccaggaaatt
721 gctttgaagt tgggttgtct agaaatacgg cgatcatact gggagatgcg gggcaatgca
781 ctagaaaaga agtctaacta tgaagtatta gaaaaagatg ttggtttaaa gcgatttttt
841 cctaagagtt tactggattc tgtcaaggcc aaaacactaa gaaaactgat ccaacaaaca
901 tttagacaat ttgccaacct taatagagaa gaaagtattc tgaaattctt tgagatcctg
961 tctccagtct acagatttga taaggaatgc ttcaagtgtg ctcttggttc aagctggatt
> 1021 atttcagtgg aactggcaat cggcccagaa gaaggaatca gttacctaac ggacaagggc
> 1081 tgcaatccca cacatcttgc tgacttcact caagtgcaaa ccattcagta ttcaaacagt
> 1141 gaagacaagg acagaaaagg aatgctacaa ctaaaaatag caggtgcacc cgagcctctg
> 1201 acagtgacgg caccatccct aaccattgcg gagaatatgg ctgacctaat agatgggtac
> 1261 tgccggctgg tgaatggaac ctcgcagtca tttatcatca gacctcagaa agaaggtgaa
> 1321 cgggctttgc catcaatacc aaagttggcc aacagcgaaa agcaaggcat gcggacacac
> 1381 gccgtctctg tgtcagaaac agatgattat gctgagatta tagatgaaga agatacttac
> 1441 accatgccct caaccaggga ttatgagatt caaagagaaa gaatagaact tggacgatgt
> 1501 attggagaag gccaatttgg agatgtacat caaggcattt atatgagtcc agagaatcca
> 1561 gctttggcgg ttgcaattaa aacatgtaaa aactgtactt cggacagcgt gagagagaaa
> 1621 tttcttcaag aagccttaac aatgcgtcag tttgaccatc ctcatattgt gaagctgatt
> 1681 ggagtcatca cagagaatcc tgtctggata atcatggagc tgtgcacact tggagagctg
> 1741 aggtcatttt tgcaagtaag gaaatacagt ttggatctag catctttgat cctgtatgcc
> 1801 tatcagctta gtacagctct tgcatatcta gagagcaaaa gatttgtaca cagggacatt
> 1861 gctgctcgga atgttctggt gtcctcaaat gattgtgtaa aattaggaga ctttggatta
> 1921 tcccgatata tggaagatag tacttactac aaagcttcca aaggaaaatt gcctattaaa
> 1981 tggatggctc cagagtcaat caattttcga cgttttacct cagctagtga cgtatggatg
> 2041 tttggtgtgt gtatgtggga gatactgatg catggtgtga agccttttca aggagtgaag
> 2101 aacaatgatg taatcggtcg aattgaaaat ggggaaagat taccaatgcc tccaaattgt
> 2161 cctcctaccc tctacagcct tatgacgaaa tgctgggcct atgaccccag caggcggccc
> 2221 aggtttactg aacttaaagc tcagctcagc acaatcctgg aggaagagaa ggctcagcaa
> 2281 gaagagcgca tgaggatgga gtccagaaga caggccacag tgtcctggga ctccggaggg
> 2341 tctgatgaag caccgcccaa gcccagcaga ccgggttatc ccagtccgag gtccagcgaa
> 2401 ggattttatc ccagcccaca gcacatggta caaaccaatc attaccaggt ttctggctac
> 2461 cctggttcac atggaatcac agccatggct ggcagcatct atccaggtca ggcatctctt
> 2521 ttggaccaaa cagattcatg gaatcataga cctcaggaga tagcaatgtg gcagcccaat
> 2581 gtggaggact ctacagtatt ggacctgcga gggattgggc aagtgttgcc aacccatctg
> 2641 atggaagagc gtctaatccg acagcaacag gaaatggaag aagatcagcg ctggctggaa
> 2701 aaagaggaaa gatttctgaa acctgatgtg agactctctc gaggcagtat tgacagggag
> 2761 gatggaagtc ttcagggtcc gattggaaac caacatatat atcagcctgt gggtaaacca
> 2821 gatcctgcag ctccaccaaa gaaaccgcct cgccctggag ctcccggtca tctgggaagc
> 2881 cttgccagcc tcagcagccc tgctgacagc tacaacgagg gtgtcaagct tcagccccag
> 2941 gaaatcagcc cccctcctac tgccaacctg gaccggtcga atgataaggt gtacgagaat
> 3001 gtgacgggcc tggtgaaagc tgtcatcgag atgtccagta aaatccagcc agccccacca
> 3061 gaggagtatg tccctatggt gaaggaagtc ggcttggccc tgaggacatt attggccact
> 3121 gtggatgaga ccattcccct cctaccagcc agcacccacc gagagattga gatggcacag
> 3181 aagctattga actctgacct gggtgagctc atcaacaaga tgaaactggc ccagcagtat
> 3241 gtcatgacca gcctccagca agagtacaaa aagcaaatgc tgactgctgc tcacgccctg
> 3301 gctgtggatg ccaaaaactt actcgatgtc attgaccaag caagactgaa aatgcttggg
> 3361 cagacgagac cacactgagc ctcccctagg agcacgtctt gctaccctct tttgaagatg
> 3421 ttctctagcc ttccaccagc agcgaggaat taaccctgtg tcctcagtcg ccagcactta
> 3481 cagctccaac ttttttgaat gaccatctgg ttgaaaaatc tttctcatat aagtttaacc
> 3541 acactttgat ttgggttcat tttttgtttt gtttttttca atcatgatat tcagaaaaat
> 其中我要研究的多态性序列为GAATCATAGA CCTCAGGAGA,等位基因为该图第2551位点的C(既C/T),所以需要针对该多态性序列设计一对引物并合成。pcr后的基因片段最好是300-500之间。我已经试过两对了，都没有成功，因为特异性太差。所以我觉得排除同源序列很重要。
e-mail : bangbanglu@163.com
