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        请教PCR

        丁香园论坛

        2025
        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’
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