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救助!!引物设计,急!!

丁香园论坛

1229
小妹从文献上找了一对引物,各种条件几乎都摸了,都出现二聚体,P不出来,请各路大哥指点迷津,引物是否可用。引物序列如下
上游序列:5’-TGA CCC ACT TGC CAC CCG TGC-3’
> > 下游序列:5’-GCA GCA CCC CAG GGC TGG C-3’
酶切位点为825,,PCR产物268bp。
基因序列为 1 ccacaatagg ggcagacctg tccatccttc tctgtgggtc ccctgtacct ttctccccca
61 acaggatcag acccagaggc agctggttgg ggtttgtcga gaagaaggat tatccagatc
121 agtcctttct aatctcagct cctgcctgta ccctcccata ctcaccaaac cctcttcccc
181 accaccctga gctgaggagc acagtttgag gtactgacaa tggggccatc tatcaccctt
241 atatcccacc tcctggcctg gttgctaagt ggccctgact gccaagatca tcaccatgga
301 tgggggccgg gaccaggggg gccactggag ctgtagttgt ctgctccttt gcacccctcc
361 caaaccccaa cccttaatcc tcacagcttt gctctaatcc tgtggggcct gatgctctta
421 tctctgcctg cagagactga ccagggaagc accccttcac cccctcctct ctcagggctt
481 gtggtggggg ctgcccctct ctatggcccc acactcctgc ccccaaccct cctcccggct
541 gggcccaagt ctctctggaa tccgtacacc tccctccctc cccccacccc ttccccccac
601 ccctgccagc tgatttcatt tgcctgacgt tgtggttggc tctacccttc cctgtcaggc
661 ccccccaacc ccccgccggt cggggccagg ccaggccagg ccagctcctc tggcagcaga
721 gcctgggcag gtgacgggcg ggcgcgggcg tcgcagctga gggagtaagg aggctcccag
781 gaaccggagc tggaaacccg gccgaggtcc agccagagcc caagtaagag ggagctggcc
841 tgggctctgc tctcctgggc tggggcgcag gcagggctca ggcaggctgg ggctggacgc
901 aaaggacctc aaacttggag ggcctagctg gggagagggt gcagccaccc agtcgtttcc
961 ctgtgtcttg gagtctggtg tgggcacgaa gggcagaagc acagcctggt ggggggttcc
1021 tcaacaccga ccccatgttc ctggcaggag ccagagtgac ccctcgacct gtcagccatg
1081 ggggagatgg agcaactgcg tcaggaagcg gagcagctca agaagcagat tgcagtaact
1141 ccagagccct acccctgggg ccccagaaaa cagctgggga catgaggagc gtggcccagg
1201 gggagggggc tgggtttgct cttgagtgac tagaagtgac cagggacttt ctaaacttgg
1261 ttcgcatatt tgagaccccc aagccccaga gccccaagac caacctgccc tgaaggccca
1321 tgcacctgcc acccccacct cgcccttgct cccctgatgt ctgctttccc tgcaggatgc
1381 caggaaagcc tgtgctgacg ttactctggc agaggtaaga ccccctgtcc cccggaaggc
1441 agggcatggg gggaggggaa gggagctccc cgacccgcaa tagcgcgttg ctccgagcat
1501 tagtacagca cgtccttgga gtgaggaaac ccattaatta attcattcga ccaacatttt
1561 agcgggacct gctctgagcc aggcaccata ttggatgcga aagatgcgga cacggttcct
1621 attctcatcc gtagaacaag agtgaccaca gccccctccc agggccttga agatgaagaa
1681 gcgatagtac tttgtaaatt ataaagccct tgtagactct agctgctagt tttctatgct
1741 gtattttgtc aattctaaga tgcacatttc ttcacatttt gacatctctg aaatcaaggt
1801 gcattcagca atggatcatt gtttcaatta gaattgacag catcttttct ttcttagtga
1861 tgcatcttag attggattaa atatgttaat agtgaacact ttttatgagc cagatactgg
1921 gctaagctgc ataaatacct gatttcattg aattttcatg atatctctgt aaggtaggtg
1981 ctatgataag cctcatttta caccttgagt aaacagactc agggaggtga agcaacttgc
2041 ccaagataac aaagcaggcc gggcgcagtg gttcacgcct gtaatcccag ccctttggga
2101 gactgacgtg aaaggatcac ttgagctcag gagtttgaga ccagcctggg caacatagtg
2161 agatctcttc ttaatataaa aaaaattgct tttaactttt taaaaaaaga tagcaatgca
2221 agtgatggca aatcaagtct gttcactctg gacccccagc ctaagtctaa aatgtggtta
2281 gagctcaagg ggcatagtac cacagatttc aggaggtaca gttcagggtc atggtgatgc
2341 cccaggcacc cagttaaaaa gctagccgtg ctgcagcggt gggtgcaagc aggctcctct
2401 cggcttggga gagccagctc tgtgcatctc ttcccaaatc tgtttccaat gacctcacat
2461 tggcagctgg aaactggtca aagtgcaagt attcgtatcg tggaaatcac cttcccacct
2521 tcctcaccaa cactggagag gtgcttgtta aacacttaca agcaacacac ctctggccat
2581 gcatgcatct ctttcccaca tcagtctaag acactccagg gcagagactt atttctctaa
2641 ggctgggggc ctgacctaga gcctggcaca gagcgggaac ttgtatagat cagtggcagt
2701 gacagacatc tgggcacgta acctgctcac cctgatattc agtgcccctc tctctgcagc
2761 tggtgtctgg cctagaggtg gtgggacgag tccagatgcg gacgcggcgg acgttaaggg
2821 gacacctggc caagatttac gccatgcact gggccactga ttctaagtga ggcttggggg
2881 ggaaccgaga atgggagggt gagagcggga gtgaggaagg cgggaagggg aggcttgcat
2941 gatatgggat gccctctccc caggctgctg gtaagtgcct cgcaagatgg gaagctgatc
3001 gtgtgggaca gctacaccac caacaaggta ccagccctgc ctccctgagc ctccaccact
3061 gcatccttcc taagggcgcc atgcctaccc tcctgtgccc agctgggagc ttggctccgg
3121 tcccatctct gctcaaacca ccctccctgc aggtgcacgc catcccactg cgctcctcct
3181 gggtcatgac ctgtgcctat gccccatcag ggaactttgt ggcatgtggg gggctggaca
3241 acatgtgttc catctacaac ctcaaatccc gtgagggcaa tgtcaaggtc agccgggagc
3301 tttctgctca cacaggtgag ggagagaccc tctcctcccc tcctgagggg ttcagggaac
3361 cctgggcttc cagtgggctg tggctctgca gccagggcac tgtccttcta accgcctcca
3421 ggttatctct cctgctgccg cttcctggat gacaacaata ttgtgaccag ctcgggggac
3481 accacgtggt gaggctgaac attgctggtg ctggggcttg ggagtgggcc cggcctttct
3541 ctaacagtct ccctccattt tggcagtgcc ttgtgggaca ttgagactgg gcagcagaag
3601 actgtatttg tgggacacac gggtgactgc atgagcctgg ctgtgtctcc tgacttcaat
3661 ctcttcattt cgggggcctg tgatgccagt gccaagctct gggatgtgcg agaggggacc
3721 tgccgtcaga ctttcactgg ccacgagtcg gacatcaacg ccatctgtgt gagtgcaccc
3781 cccaccccag cttcactcca actccttccc cgacactccc cacaacacat acaatacaca
3841 tcctctgccc ctcccatagc ttccctagcc ctttcttact gtattttttt tttttttttt
3901 tttttttttg agacagagtc tcactttgtc gcccaggctg gagtgcagtg gtgcaatctt
3961 ggctcactgc aacctctgcc tcctgggttg gagcaattct cctgcttcag cctcctgagt
4021 agctgggatt acaggtgtgc gccaccatgc ccggctaatt tttgtatttt tagtagagat
4081 ggggttttgc catgttggcc aggctggtct tgaactcttg acctgaggtg atcctcccac
4141 ctcagcttcc cagagtgcag tgctgggatt acagatgtga gccactgtgc ccagtcttcc
4201 ctagcccttt ctttctttcc tttttttgga gacagagtct cacactgttg cccagtctgg
4261 atgcagtggt gctatcatag ctcaccgcag tctcaacctc cagggttcaa ccaatcctcc
4321 caccacagcc tcctgagtag ctgggactac agatgcgtgt gaccacgctt ggctaatgtt
4381 ttgatttttg tgtagagatg ggttcccact atgttgccca ggctgagctg gaacttctga
4441 gctcaagcga tcctcctgcc tcagtctccc aaaatattgg gattacaggc atgagccacc
4501 acgcctagcc cattttatca cgtttgcttt atcatttttt ctctgtagag aaatagccat
4561 agatacacac acacatacac acttagatgt agatatacat tttctttctg tgtcttttga
4621 aagtaggttg cagacatcat gtttctttac cccttaagta cttcccgaaa caaagccctt
4681 ctgtagcata atcactgtat gattcttaag atcaggaaac tcaacattga cacaatacta
4741 cagtctacag tctatattca aatttcacca atagcctcaa aaatgtccat aatagctcat
4801 gtttttttct gtccaggatc caatccagaa acgtgcatta cctttcctcg ccatatcttt
4861 ttcgtcacca cctttaaact ggaactgttt tcccaccttt gtcttttatg tcattgacgt
4921 ttttgaagag tacaaaccag ttttctgtag actctccctc cgtttgagct tatctgacat
4981 ttgctcgccg tgagatccag gccttgcatt tgtactggac cctgttctta cacaccctga
5041 tccagcccac ttgtgtagtc tgggagtctg ggacaacctc cgtccgccct tctagccggg
5101 tcactgcagg caagccttgg tgctcttgcc tgcgacgtgg aaatgatgcc tgcctgcagc
5161 gctgtatagt gcagagcggg cgaggggcat agggaagtca ctggcacgtg gtatgtgttg
5221 gcagggctgc ttctcacccc aaaccaaggg agggacaggc agggaggctg agagcagcgg
5281 cttgccctgg agctgtcagg tgggaggcag agggcgggag aggctgtggg ctgcccaggt
5341 ctgatccctg acccacttgc cacccgtgcc ctcagttctt ccccaatgga gaggccatct
5401 gcacgggctc ggatgacgct tcctgccgct tgtttgacct gcgggcagac caggagctga
5461 tctgcttctc ccacgagagc atcatctgcg gcatcacgtc cgtggccttc tccctcagtg
5521 gccgcctact attcgctggc tacgacgact tcaactgcaa tgtctgggac tccatgaagt
5581 ctgagcgtgt gggtaagggc cagccctggc tgctgcttcc tcagctggaa ggaccctccc
5641 cagccctccc tccccattct gtacccccca tcagctccca tttcggactc tcttactgct
5701 gtcccttgtc actgggtgac tccacccctg gaatccagta ccccttggtt cccaactagg
5761 actgttttcc ctcagtgttg ctctaagcag cctctctcca ctgcccaatg ccatgactgc
5821 tccctgccct aggagatctg tggaccatga ctgtccagtc agttctgggt tcctggcatt
5881 tcaggggcac ccactgagag gcaagacagc ctcagggaaa catggaatca aggcagaatc
5941 aaggagatct ggagtggccc gagggccctg actgcagcct agggctcatc taagactagc
6001 ctgaagttgg tcagatagga tggcttcttc tctatcaaga ggccaggtgc tgatgaaaat
6061 aacactggcc aggggcagaa tccaaatcct aattctgttg ctcactttct gagtgtcctt
6121 gcacaagtca ctcctccatg ggcctcagtt tcttcatctg gaagcaaaga aataggatga
6181 acggttgcct tctcttttcc agcacctgtc aaggtgccct cgggtttttt catagtacac
6241 agatgcttcc agcctttctc tgtctgatcc cagccctccc ccaaccaaag ctcttgacag
6301 agaaccccct gccctacagc taatcctctt tcagtctctt agccttcttc aggggttgag
6361 cccagtctac cgagtctagg atccctgcat gcatgtgctc aagcacacat gcacacacac
6421 acgtacacac acccacacat gcatacacat gcacacatat ccccccacac acccacatac
6481 acacacacac ccacacaccc acacatacac ttacacgcat gcacacactg ctttccaaat
6541 cagcttggag agacaggctg actcctttcc ctcttcctca ggcatcctct ctggccacga
6601 taacagggtg agctgcctgg gagtcacagc tgacgggatg gctgtggcca caggttcctg
6661 ggacagcttc ctcaaaatct ggaactgagg aggctggaga aagggaagtg gaaggcagtg
6721 aacacactca gcagccccct gcccgacccc atctcattca ggtgttctct tctatattcc
6781 gggtgccatt cccactaagc tttctccttt gagggcagtg gggagcatgg gactgtgcct
6841 ttgggaggca gcatcaggga cacaggggca aagaactgcc ccatctcctc ccatggcctt
6901 ccctccccac agtcctcaca gcctctccct taatgagcaa ggacaacctg cccctcccca
6961 gccctttgca ggcccagcag acttgagtct gaggccccag gccctaggat tcctccccca
7021 gagccactac ctttgtccag gcctgggtgg tatagggcgt ttggccctgt gactatggct
7081 ctggcaccac tagggtcctg gccctcttct tattcatgct ttctcctttt tctacctttt
7141 tttctctcct aagacacctg caataaagtg tagcaccctg gta

我用Primer5.0软件设计,不知该用哪段基因序列去设计引物,也找不到和文献一致的引物,急切求助各位大哥,不胜感激!
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