请教PCR
丁香园论坛
1866
我想请教大家：我的PCR跑出四条带，亮度不相上下，包括目的基因，其余杂带比目的带短，退火温度52-58都试过，没改善。接着调高退火温度有可能得到特异条带吗？会不会引物设计有问题？我用的是PRIMER DESIGNER 1.01.烦劳看看我的基因序列，引物，要真有问题，就死心塌地重订引物了。
1 cggcagcagc gggaggcgag gggcgccacc gaggagccga gcccgccgag ccgggcgctc
61 tatttcagcg ggcgaggcga gcagctgcga gtcctccggg ccgacctcga gctgccccgg
121 gacgcgttca cgctgcaagt gtggctgcga gcggaggggg gccagaggtc tccggcagtg
181 atcacagggc tgtatgacaa atgttcttat atctcacgtg accgaggatg ggtcgtgggc
241 attcacacca tcagtgacca agacaacaaa gacccacgct actttttctc cttgaagaca
301 gaccgagccc ggcaagtgac caccatcaat gcccaccgca gctacctccc aggccagtgg
361 gtatacctag ctgccaccta tgatgggcag ttcatgaagc tctatgtgaa tggtgcccag
421 gtggccacct ctggggaaca agtgggtggc atattcagcc cactgaccca gaagtgcaaa
481 gtgctcatgt tagggggcag tgccctgaat cacaactacc ggggctacat cgagcacttc
541 agtctgtgga aggtggccag gactcagcgg gagatactgt ctgacatgga aacccatggc
601 gcccacactg ctctacctca gctcctcctc caggagaact gggacaatgt gaagcatgcc
661 tggtccccca tgaaggatgg cagcagcccc aaagtggaat tcagcaatgc ccacggcttt
721 ctgctggaca cgagtctgga gcctcctctg tgcggacaga cattgtgtga caacacagag
781 gtcattgcca gctacaatca gctctcaagt ttccgccagc ccaaggtggt gcgctaccgc
841 gtggtcaacc tctatgaaga tgatcataag aacccgacgg tgacgcgcga gcaggtggac
901 ttccagcacc atcagctggc tgaggccttc aagcaataca acatctcctg ggagctggac
961 gtgctggagg tgagcaactc ctcccttcgc cgccgcctca tcctggccaa ctgtgacatc
1021 agcaagattg gggatgagaa ctgtgacccc gagtgcaacc acacgctgac gggccacgac
1081 ggcggggatt gccgccacct gcgccaccct gccttcgtga agaagcagca caacggggtg
1141 tgtgacatgg actgcaacta tgaacggttc aactttgatg gtggagagtg ctgtgaccct
1201 gaaatcacca atgtcactca gacttgcttt gaccccgact ctccacacag agcctacttg
1261 gatgttaatg agctgaagaa cattcttaaa ttggatggat caacacatct caatattttc
1321 tttgcaaaat cctcagagga ggagttggca ggagtagcaa cttggccatg ggacaaggag
1381 gccctgatgc acttaggtgg cattgtcttg aacccatctt tctatggcat gcctgggcac
1441 acccacacca tgatccatga gattggtcac agcctgggcc tctatcacgt cttccgaggc
1501 atctcagaaa tccagtcctg cagtgacccc tgcatggaga cagagccctc cttcgagact
1561 ggagacctct gcaatgatac caacccagcc cctaaacaca agtcctgtgg tgacccaggg
1621 ccaggaaatg acacctgtgg ctttcatagc ttcttcaaca ctccttacaa caacttcatg
1681 agctatgcag atgacgactg tacggactcc ttcacgccca atcaagtcgc cagaatgcac
1741 tgttacctgg acctggtcta ccagggctgg cagccctcca ggaaaccagc gcctgttgcc
1801 ctcgcccccc aagttctggg ccacacaacg gactctgtga cactggagtg gttcccacct
1861 atagatggcc atttctttga aagagaattg ggatcagcat gtcatctttg cctggaaggg
1921 agaatcctgg tgcagtatgc ttccaacgct tcctccccaa tgccctgcag cccatcagga
1981 cactggagcc ctcgtgaagc agaaggtcat cctgatgttg aacagccctg taagtccagt
2041 gtccgcacct ggagcccaaa ttcagctgtc aacccacaca cggttcctcc agcctgccct
2101 gagcctcaag gctgctacct cgagctggag ttcctctacc ccttggtccc tgagtctctg
2161 accatttggg tgacctttgt ctccactgac tgggactcta gtggagctgt caatgacatc
2221 aaactgttgg ctgtcagtgg gaagaacatc tccctgggtc ctcagaatgt cttctgtgat
2281 gtcccactga ccatcagact ctgggacgtg ggcgaggagg tgtatggcat ccaaatctac
2341 acgctggatg agcacctgga gatcgatgct gccatgttga cctccactgc agacacccca
2401 ctctgtctac agtgtaagcc cctgaagtat aaggtggtcc gggaccctcc tctccagatg
2461 gatgtggcct ccatcctaca tctcaatagg aaattcgtag acatggatct aaatcttggc
2521 agtgtgtacc agtattgggt cataactatt tcaggaactg aagagagtga gccatcacct
2581 gctgtcacat acatccatgg acgtgggtac tgtggcgatg gcattataca aaaagaccaa
2641 ggtgaacaat gcgacgacat gaataagatc aatggtgatg gctgctccct tttctgccga
2701 caagaagtct ccttcaattg tattgatgaa cccagccggt gctatttcca tgatggtgat
2761 ggggtatgtg aggagtttga acaaaaaacc agcattaagg actgtggtgt ctacacgccc
2821 cagggattcc tggatcagtg ggcatccaat gcttcagtat ctcatcaaga ccagcaatgc
2881 ccaggctggg tcatcatcgg acagccagca gcatcccagg tgtgtcgaac caaggtgata
2941 gatctcagtg aaggcatttc ccagcatgcc tggtaccctt gcaccatcag ctacccatat
3001 tcccagctgg ctcagaccac tttttggctc cgggcgtatt tttctcaacc aatggttgcc
3061 gcagctgtca ttgtccacct ggtgacggat gggacatatt atggggacca aaagcaggag
3121 accatcagcg tgcagctgct tgataccaaa gatcagagcc acgatctagg cctccatgtc
3181 ctgagctgca ggaacaatcc cctgattatc cctgtggtcc atgacctcag ccagcccttc
3241 taccacagcc aggcggtacg tgtgagcttc agttcgcccc tggtcgccat ctcgggggtg
3301 gccctccgtt ccttcgacaa ctttgacccc gtcaccctga gcagctgcca gagaggggag
3361 acctacagcc ctgccgagca gagctgcgtg cacttcgcat gtgagaaaac tgactgtcca
3421 gagctggctg tggagaatgc ttctctcaat tgctccagca gcgaccgcta ccacggtgcc
3481 cagtgtactg tgagctgccg gacaggctac gtgctccaga tacggcggga tgatgagctg
3541 atcaagagcc agacgggacc cagcgtcaca gtgacctgta cagagggcaa gtggaataag
3601 caggtggcct gtgagccagt cgactgcagc atcccagatc accatcaagt ctatgctgcc
3661 tccttctcct gccctgaggg caccaccttt ggcagtcaat gttccttcca gtgccgtcac
3721 cctgcacaat tgaaaggcaa caacagcctc ctgacctgca tggaggatgg gctgtggtcc
3781 ttcccagagg ccctgtgtga gctcatgtgc ctcgctccac cccctgtgcc caatgcagac
3841 ctccagaccg cccggtgccg agagaataag cacaaggtgg gctccttctg caaatacaaa
3901 tgcaagcctg gataccatgt gcctggatcc tctcggaagt caaagaaacg ggccttcaag
3961 actcagtgta cccaggatgg cagctggcag gagggagctt gtgttcctgt gacctgtgac
4021 ccacctccac caaaattcca tgggctctac cagtgtacta atggcttcca gttcaacagt
4081 gagtgtagga tcaagtgtga agacagtgat gcctcccagg gacttgggag caatgtcatt
4141 cattgccgga aagatggcac ctggaacggc tccttccatg tctgccagga gatgcaaggc
4201 cagtgctcgg ttccaaacga gctcaacagc aacctcaaac tgcagtgccc tgatggctat
4261 gccatagggt cggagtgtgc cacctcgtgc ctggaccaca acagcgagtc catcatcctg
4321 ccaatgaacg tgaccgtgcg tgacatcccc cactggctga accccacacg ggtagagaga
4381 gttgtctgca ctgctggtct caagtggtat cctcaccctg ctctgattca ctgtgtcaaa
4441 ggctgtgagc ccttcatggg agacaattat tgtgatgcca tcaacaaccg agccttttgc
4501 aactatgacg gtggggattg ctgcacctcc acagtgaaga ccaaaaaggt caccccattc
4561 cctatgtcct gtgacctaca aggtgactgt gcttgtcggg acccccaggc ccaagaacac
4621 agccggaaag acctccgggg atacagccat ggctaaggaa ggacaagaag ttgtcaaaga
4681 attcccaacg ccaggaccca catccctttg gtattgattt cacagtcagc tgctcaacgg
4741 aatggcctct ccacaccagg gatccttagc acccaaccgg tctgccttta attttaccca
4801 ggaaggactc acattggggc gaatgaacca agtttcgcca tgctggatga tgaaatggat
4861 tcccatccca aagtctgaga tggattgcat atacagtgtg cagtcccaga gcctcctaaa
4921 attctagcca tttgtcacac aaccacagca aaaaaaaa
PAPP-A: 上游5’－AGCTTGTGTTCCTGTGACCT-3’
下游5’－GATACCACTTGAGACCAGCA-3’
1 cggcagcagc gggaggcgag gggcgccacc gaggagccga gcccgccgag ccgggcgctc
61 tatttcagcg ggcgaggcga gcagctgcga gtcctccggg ccgacctcga gctgccccgg
121 gacgcgttca cgctgcaagt gtggctgcga gcggaggggg gccagaggtc tccggcagtg
181 atcacagggc tgtatgacaa atgttcttat atctcacgtg accgaggatg ggtcgtgggc
241 attcacacca tcagtgacca agacaacaaa gacccacgct actttttctc cttgaagaca
301 gaccgagccc ggcaagtgac caccatcaat gcccaccgca gctacctccc aggccagtgg
361 gtatacctag ctgccaccta tgatgggcag ttcatgaagc tctatgtgaa tggtgcccag
421 gtggccacct ctggggaaca agtgggtggc atattcagcc cactgaccca gaagtgcaaa
481 gtgctcatgt tagggggcag tgccctgaat cacaactacc ggggctacat cgagcacttc
541 agtctgtgga aggtggccag gactcagcgg gagatactgt ctgacatgga aacccatggc
601 gcccacactg ctctacctca gctcctcctc caggagaact gggacaatgt gaagcatgcc
661 tggtccccca tgaaggatgg cagcagcccc aaagtggaat tcagcaatgc ccacggcttt
721 ctgctggaca cgagtctgga gcctcctctg tgcggacaga cattgtgtga caacacagag
781 gtcattgcca gctacaatca gctctcaagt ttccgccagc ccaaggtggt gcgctaccgc
841 gtggtcaacc tctatgaaga tgatcataag aacccgacgg tgacgcgcga gcaggtggac
901 ttccagcacc atcagctggc tgaggccttc aagcaataca acatctcctg ggagctggac
961 gtgctggagg tgagcaactc ctcccttcgc cgccgcctca tcctggccaa ctgtgacatc
1021 agcaagattg gggatgagaa ctgtgacccc gagtgcaacc acacgctgac gggccacgac
1081 ggcggggatt gccgccacct gcgccaccct gccttcgtga agaagcagca caacggggtg
1141 tgtgacatgg actgcaacta tgaacggttc aactttgatg gtggagagtg ctgtgaccct
1201 gaaatcacca atgtcactca gacttgcttt gaccccgact ctccacacag agcctacttg
1261 gatgttaatg agctgaagaa cattcttaaa ttggatggat caacacatct caatattttc
1321 tttgcaaaat cctcagagga ggagttggca ggagtagcaa cttggccatg ggacaaggag
1381 gccctgatgc acttaggtgg cattgtcttg aacccatctt tctatggcat gcctgggcac
1441 acccacacca tgatccatga gattggtcac agcctgggcc tctatcacgt cttccgaggc
1501 atctcagaaa tccagtcctg cagtgacccc tgcatggaga cagagccctc cttcgagact
1561 ggagacctct gcaatgatac caacccagcc cctaaacaca agtcctgtgg tgacccaggg
1621 ccaggaaatg acacctgtgg ctttcatagc ttcttcaaca ctccttacaa caacttcatg
1681 agctatgcag atgacgactg tacggactcc ttcacgccca atcaagtcgc cagaatgcac
1741 tgttacctgg acctggtcta ccagggctgg cagccctcca ggaaaccagc gcctgttgcc
1801 ctcgcccccc aagttctggg ccacacaacg gactctgtga cactggagtg gttcccacct
1861 atagatggcc atttctttga aagagaattg ggatcagcat gtcatctttg cctggaaggg
1921 agaatcctgg tgcagtatgc ttccaacgct tcctccccaa tgccctgcag cccatcagga
1981 cactggagcc ctcgtgaagc agaaggtcat cctgatgttg aacagccctg taagtccagt
2041 gtccgcacct ggagcccaaa ttcagctgtc aacccacaca cggttcctcc agcctgccct
2101 gagcctcaag gctgctacct cgagctggag ttcctctacc ccttggtccc tgagtctctg
2161 accatttggg tgacctttgt ctccactgac tgggactcta gtggagctgt caatgacatc
2221 aaactgttgg ctgtcagtgg gaagaacatc tccctgggtc ctcagaatgt cttctgtgat
2281 gtcccactga ccatcagact ctgggacgtg ggcgaggagg tgtatggcat ccaaatctac
2341 acgctggatg agcacctgga gatcgatgct gccatgttga cctccactgc agacacccca
2401 ctctgtctac agtgtaagcc cctgaagtat aaggtggtcc gggaccctcc tctccagatg
2461 gatgtggcct ccatcctaca tctcaatagg aaattcgtag acatggatct aaatcttggc
2521 agtgtgtacc agtattgggt cataactatt tcaggaactg aagagagtga gccatcacct
2581 gctgtcacat acatccatgg acgtgggtac tgtggcgatg gcattataca aaaagaccaa
2641 ggtgaacaat gcgacgacat gaataagatc aatggtgatg gctgctccct tttctgccga
2701 caagaagtct ccttcaattg tattgatgaa cccagccggt gctatttcca tgatggtgat
2761 ggggtatgtg aggagtttga acaaaaaacc agcattaagg actgtggtgt ctacacgccc
2821 cagggattcc tggatcagtg ggcatccaat gcttcagtat ctcatcaaga ccagcaatgc
2881 ccaggctggg tcatcatcgg acagccagca gcatcccagg tgtgtcgaac caaggtgata
2941 gatctcagtg aaggcatttc ccagcatgcc tggtaccctt gcaccatcag ctacccatat
3001 tcccagctgg ctcagaccac tttttggctc cgggcgtatt tttctcaacc aatggttgcc
3061 gcagctgtca ttgtccacct ggtgacggat gggacatatt atggggacca aaagcaggag
3121 accatcagcg tgcagctgct tgataccaaa gatcagagcc acgatctagg cctccatgtc
3181 ctgagctgca ggaacaatcc cctgattatc cctgtggtcc atgacctcag ccagcccttc
3241 taccacagcc aggcggtacg tgtgagcttc agttcgcccc tggtcgccat ctcgggggtg
3301 gccctccgtt ccttcgacaa ctttgacccc gtcaccctga gcagctgcca gagaggggag
3361 acctacagcc ctgccgagca gagctgcgtg cacttcgcat gtgagaaaac tgactgtcca
3421 gagctggctg tggagaatgc ttctctcaat tgctccagca gcgaccgcta ccacggtgcc
3481 cagtgtactg tgagctgccg gacaggctac gtgctccaga tacggcggga tgatgagctg
3541 atcaagagcc agacgggacc cagcgtcaca gtgacctgta cagagggcaa gtggaataag
3601 caggtggcct gtgagccagt cgactgcagc atcccagatc accatcaagt ctatgctgcc
3661 tccttctcct gccctgaggg caccaccttt ggcagtcaat gttccttcca gtgccgtcac
3721 cctgcacaat tgaaaggcaa caacagcctc ctgacctgca tggaggatgg gctgtggtcc
3781 ttcccagagg ccctgtgtga gctcatgtgc ctcgctccac cccctgtgcc caatgcagac
3841 ctccagaccg cccggtgccg agagaataag cacaaggtgg gctccttctg caaatacaaa
3901 tgcaagcctg gataccatgt gcctggatcc tctcggaagt caaagaaacg ggccttcaag
3961 actcagtgta cccaggatgg cagctggcag gagggagctt gtgttcctgt gacctgtgac
4021 ccacctccac caaaattcca tgggctctac cagtgtacta atggcttcca gttcaacagt
4081 gagtgtagga tcaagtgtga agacagtgat gcctcccagg gacttgggag caatgtcatt
4141 cattgccgga aagatggcac ctggaacggc tccttccatg tctgccagga gatgcaaggc
4201 cagtgctcgg ttccaaacga gctcaacagc aacctcaaac tgcagtgccc tgatggctat
4261 gccatagggt cggagtgtgc cacctcgtgc ctggaccaca acagcgagtc catcatcctg
4321 ccaatgaacg tgaccgtgcg tgacatcccc cactggctga accccacacg ggtagagaga
4381 gttgtctgca ctgctggtct caagtggtat cctcaccctg ctctgattca ctgtgtcaaa
4441 ggctgtgagc ccttcatggg agacaattat tgtgatgcca tcaacaaccg agccttttgc
4501 aactatgacg gtggggattg ctgcacctcc acagtgaaga ccaaaaaggt caccccattc
4561 cctatgtcct gtgacctaca aggtgactgt gcttgtcggg acccccaggc ccaagaacac
4621 agccggaaag acctccgggg atacagccat ggctaaggaa ggacaagaag ttgtcaaaga
4681 attcccaacg ccaggaccca catccctttg gtattgattt cacagtcagc tgctcaacgg
4741 aatggcctct ccacaccagg gatccttagc acccaaccgg tctgccttta attttaccca
4801 ggaaggactc acattggggc gaatgaacca agtttcgcca tgctggatga tgaaatggat
4861 tcccatccca aagtctgaga tggattgcat atacagtgtg cagtcccaga gcctcctaaa
4921 attctagcca tttgtcacac aaccacagca aaaaaaaa
PAPP-A: 上游5’－AGCTTGTGTTCCTGTGACCT-3’
下游5’－GATACCACTTGAGACCAGCA-3’
