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        RT-PCR引物设计,请各位多多指教!!

        丁香园论坛

        1660
        我依据所查的资料,用primer.0设计了以下引物,
        上游:TGGGACACTGATGAGGGATA 下游:GGCACAAGGCAGACAAAT
        其中下游链存在错配,这是否会影响扩增结果。麻烦各位看看整体设计否合理??

        附:
        D63902. Reports Mus musculus mRNA...[gi:1088466] Links

        LOCUS MUSEFP 5475 bp mRNA linear ROD 29-MAY-2002
        DEFINITION Mus musculus mRNA for estrogen-responsive finger protein, complete
        cds.
        ACCESSION D63902
        VERSION D63902.1 GI:1088466
        KEYWORDS .
        SOURCE Mus musculus (house mouse)
        ORGANISM Mus musculus
        Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
        Mammalia; Eutheria; Rodentia; Sciurognathi; Muridae; Murinae; Mus.
        REFERENCE 1 (bases 1 to 5475)
        AUTHORS Orimo,A., Inoue,S., Ikeda,K., Noji,S. and Muramatsu,M.
        TITLE Molecular cloning, structure, and expression of mouse
        estrogen-responsive finger protein Efp. Co-localization with
        estrogen receptor mRNA in target organs
        JOURNAL J. Biol. Chem. 270 (41), 24406-24413 (1995)
        MEDLINE 96025835
        PUBMED 7592654
        REFERENCE 2 (bases 1 to 5475)
        AUTHORS Orimo,A.
        TITLE Direct Submission
        JOURNAL Submitted (23-AUG-1995) Akira Orimo, Saitama Medical School, 2nd
        Biochemistry; 38 Morohongo Moroyama, Iruma-gun, Saitama 350-04,
        Japan (E-mail:a orimo@ ddbj.nig.ac.jp, Tel:0492-76-1490,
        Fax:0492-94-9751)
        FEATURES Location/Qualifiers
        source 1..5475
        /organism="Mus musculus"
        /mol_type="mRNA"
        /db_xref="taxon:10090"
        /chromosome="11"
        /map="11-C"
        /tissue_type="uterus, ovary, placenta"
        /clone_lib="Zap"
        CDS 10..1914
        /codon_start=1
        /product="estrogen-responsive finger protein"
        /protein_id="BAA09941.1"
        /db_xref="GI:1088467"
        /translation="MAELNPLAEELSCSVCLELFKEPVTTPCGHNFCTSCLDETWVVQ
        GPPYRCPQCRKVYQVRPQLQKNTVMCAVVEQFLQAEQARTPVDDWTPPARFSASSAAT
        QVACDHCLTEIAVKTCLVCMASFCQEHLRPHFDSPAFQDHPLQSPIRDLLRRKCTQHN
        RLREFFCPEHGECICHICLVEHKTCSPTTLSQASADLEYKLRNKLTIMHSHINGATKA
        LEDVRSKQQCVQDSMKRKMEQLRQEYMEMKAVIDAAETSSLRRLKEEEKRVYGKFDTI
        YQVLVKKKSEMQKLKAEVELIMDKGDEFEFLEKAAKLQGESTKPVYIPKIDLDHDLIM
        GIYQGAADLKSELKHSIKKLQKKSEEHNGSGNKGDQTQSTFKPVQPSKKTIQEKKTKK
        TPVAPGPPSHFSPNKLPTFGAPGQSLDSKATSPDAAPKASAAQPDSIGVKAKVLENFL
        TKSRTELLEYFVKVIFDYNTAHNKVSLSNKYTTASVSDGLQHYRSHPQRFTYCSQVLG
        LHCYKNGIHYWEVELQKNNFCGVGICYGSMERQGPESRLGRNPNSWCVEWFNNKISAW
        HNNVEKTLPSTKATRVGVLLNCDHGFVIFFAVTEKVHLMYKFKVDFTEALYPAFWVFS
        AGTTLSICSK"
        polyA_site 5475
        ORIGIN
        1 acgagcgcga tggcggagct gaatcctctg gccgaggagc tgtcctgctc cgtctgcctg
        61 gagctcttca aggaacctgt caccaccccg tgcggccaca acttctgcac gtcatgcctg
        121 gatgagacgt gggtcgtcca gggcccgccg taccgatgcc cgcaatgtcg caaagtgtac
        181 caggtgcgcc cgcagctgca aaagaacacg gtaatgtgcg cggtggtgga gcagttcctg
        241 caggccgagc aggcgcggac cccggtggac gactggacgc ctcctgcccg cttctccgcc
        301 tccagcgcag ccacccaggt ggcctgcgac cactgcctga cggagatcgc agtgaagacg
        361 tgcctcgtgt gcatggcctc tttctgccag gagcacttga ggcctcactt cgacagtccg
        421 gccttccagg atcaccccct acagtcgccc atccgtgacc ttctgcgccg caaatgtacc
        481 cagcacaacc gccttcggga attcttttgt cccgagcatg gcgagtgcat ctgccatatc
        541 tgcttggtag agcacaagac ctgctcccct acgaccctaa gtcaagccag cgccgacctg
        601 gagtacaaac tgaggaataa actcactatc atgcacagcc acatcaatgg ggcaaccaaa
        661 gcattggagg atgtgagatc caagcaacag tgtgtgcagg attctatgaa gaggaagatg
        721 gaacagctga gacaggagta tatggaaatg aaggctgtca tcgacgcagc agagaccagc
        781 tccttgcgca ggttgaagga ggaggagaag agagtctacg gaaaattcga caccatctac
        841 caggttctcg tcaagaagaa gagtgagatg cagaaactga aggcagaggt tgagctcatt
        901 atggacaaag gggatgagtt tgagtttctg gagaaagctg caaaattgca aggagagtca
        961 acaaaaccag tctacatccc taagatagat ctggatcatg atttgataat gggaatctac
        1021 caaggggcgg cggacctcaa gagtgaactg aagcactcca ttaagaagct acagaagaag
        1081 tctgaggagc acaatggctc aggtaacaag ggagaccaga cacagtccac ttttaaaccc
        1141 gtacaacctt cgaagaagac cattcaagaa aagaagacca agaaaacccc tgttgctccc
        1201 ggccctccct cccacttctc acccaacaag ttgcccacct ttggagctcc tggccaatca
        1261 ctggattcaa aagcaacttc ccctgatgcc gcacccaaag ccagtgctgc acagccagac
        1321 tctataggag tcaaggctaa ggtgctagaa aacttcttaa ccaagtccag aacggagctt
        1381 ctggagtatt tcgtgaaagt catcttcgac tacaataccg cccacaacaa agtgtctctg
        1441 tcaaacaagt acaccaccgc ctctgtgtct gatgggctcc agcactaccg gtcccatccc
        1501 cagaggttca cctactgctc tcaggtcctg gggctgcact gctacaagaa tggcattcac
        1561 tactgggagg tggaactaca gaagaacaac ttctgcggcg taggcatctg ctacggcagc
        1621 atggaacggc agggccccga gagccggctg ggccgcaacc ccaactcatg gtgtgtggag
        1681 tggttcaata acaagatctc cgcctggcac aacaacgtgg agaagactct gccctccacc
        1741 aaggccactc gagtcggcgt gctgctcaac tgtgaccacg gcttcgtcat tttctttgct
        1801 gtcaccgaaa aggtgcacct gatgtacaag ttcaaggtgg acttcaccga agctctgtac
        1861 ccagccttct gggtgttctc ggcgggtacc accctctcta tctgctccaa gtagccaggt
        1921 ccctaggcta gccacagaca cttgggcttg ctggggaaca tccaagactg gagggaactc
        1981 tgaacttgtg agtccctatc agataagaaa ttgggagggg agctggggaa ggggtgggaa
        2041 gaatcaaagg ggaggtttag gctcagaaag gggtggggtg attgtcttgt ggatggagaa
        2101 gcatatctac ctcctcatgc ccccgagggc agggtgacct cctgccagtg ttctgggaaa
        2161 tctctccagg ctgctagtta cttgggcaga gcctcagaga gtaaaattat tagtgtacct
        2221 gttgtcttac agaaaaatct atagctgtta gtcatgttct ttctcaggtg acagatgtcc
        2281 accctattct actctctggg gtagccttca gatctttgtg ttttgattat ggcctagatt
        2341 tttgtttgtt ttaaagccag ctgtagcctt tgggaaatag aaacagacgc agcttgatga
        2401 gttcaaggac agcctgatgt atatagtggg tttttggaca gcttgtctct gaagaacaaa
        2461 aatccagttc ttgagttctg tatgtatatg tgtgtgtgtg tgtgtgtgtg tgtgtgtgta
        2521 tatatataaa atcatcctct ttggggctgg ggtctgaatc tatggccttt acccctgagc
        2581 tacatcccta ctctgaattt ccatataacc cttcccccac aaggaacctt ttgtagataa
        2641 ggctgcagcc tgtgttgatt tgtgtcaagg taacccagac ctggttgaga cggaaatctc
        2701 tccctcttca aggggcagtt ctctggggac atgttgacgg tggccatgga gttagaggct
        2761 ttcgtcccat agcctaggcc aggggtggag gttgaggaac agaaccctgg ctggattgtg
        2821 ggagccctga tggctttgct cttactatta tcttgccttg acagctgttc ccaacagtaa
        2881 tgtccccatc ctctggattg tagatactgg gctgatggct ataaatgccg ccatttgcct
        2941 tttgtggctt ctgtcacagt taagtggaca gccacaccac attcccaaag cctaccatca
        3001 tggagactgg ctgtgtgtgg ttgcttcagg aaccttgcta gatcacctat tggtggacag
        3061 cctgtggcct gaagcgagag acgagcggga gatagctgga ttcctgttta tctctagcca
        3121 cagattacag gcccagtgat gagtcagacc cgtcacctgc ccgacccttt ctgctctctt
        3181 cctcattttt gagccaattc cagatgttca tgtagaccaa actacaggtt aggaagactt
        3241 tgaaggatga ggaaacagta aataaataac aaggagagtc caggcagtgg ctgttttctc
        3301 tgctgtcttt cacacagtga aaatggcccg gccataaagg gcaaggatgg atggctgact
        3361 gggatctccc cacctcctgt cgtgatgtca aatccagaag atgtgaggat ttgacctctt
        3421 gtgacaggag tccagctctc tctttcctaa gcggggagct cctgcttgca agcattctta
        3481 tgaagagagg tcaccatagg gaaggtaggc cctagacctt ttcatttgat ggatatttga
        3541 gaacacatct caggtcttta tggaattcca ggctcttagt attggcattc ccgacagacg
        3601 tgtgatctgt gtcttctccc agaggaatat tctattccct ttaagcatct ttacagggaa
        3661 catgtccctg tagatggtat gtgggctttg aaagatttct tcctttgcca cacaaattct
        3721 tggcgatact tctccaactt tgaaggtgcc caaaaagctt ctttgctggt cttgttttga
        3781 gccttgtttg aggccagatc agacttggga aacaactagg ttcagagaag tggaacacca
        3841 aggattaact caggagtcct gcatgggctc ttctgcagaa aaggagccgg acagtcagga
        3901 atctttcccc aggcagtgtg cagctcaggc taggaaggca ttcgttccct ctgttttatg
        3961 agagacccaa gcaggcaacc aaagcccaga gctgtgcaag atgctttgga gttgtgagtg
        4021 gaggggtgta ccctttctgt ctggtacttt tctgtttgca gtgataagac cttaccttaa
        4081 ccataagcat ttctgggtag atggactaag agttcatcag agtgcacatc tcacttggct
        4141 ctttgcagag tcaggcaggt gggaaggtgg gggatggctc tgctcaagtc ctcttgtcat
        4201 tccaacatgg aagagactcc taacgtgggt gaccaagaaa accccagggc cctgtagaca
        4261 tacagaaatt tccagaaatg ttgatgtaaa atcctgtcgt tgatcaagat accggggact
        4321 gtctcagtca ctgtggtgtg ataaactaaa ggtggaatct ttgtattttt ctgatcactc
        4381 agaaattgat tttttttacc tattactaat agcgatattt tatattttct actattgatt
        4441 attaattaat tactaatgtt ttcactatga cttcttgaac ctagtgtttt tagaggtaat
        4501 tcattaagat aatcaatcat tcattgtaca gggcaaggct ggctgctgta ccttactttg
        4561 tatgcaaaca gcatccagca tcctctctgg attgaggtgt gggtgaaaga tgtcactcaa
        4621 gctgaaatcc actgtcgagg taccaccccc taagagctac tgcccagggt cactgggtgc
        4681 tcaggccctg ctccggaagc ctgctatagc gtagtcagat cacatgaaac tggataaaga
        4741 acactcaaga gagagaactt agcttcaaga cagtgccagg gtgggacact gatgagggat
        4801 aagcctgctt gagtaaccgg caagctgcag tgggaaccca gagcttgctt gaatattttt
        4861 cttgcggttg catagacttg accatttcta tcgtaagtta aatggtgaaa tggaaaagtc
        4921 ttgaccagag ataaacccta ttcatgatct tctctacata ggactctttg gctattaaag
        4981 aattacgctt gcctttgtcg ctggcgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
        5041 tgtgtgtgtg tgtgtgtgtg cgcacgcgct tgtgcttgca tgagtgcgtg tttaagttct
        5101 cggaatttcc tgagaggatg gagtgccatt gttatttgtc tgccttgtgc ctgacacagc
        5161 ggaatttatg tcaagagatg acccagcatg gtagctagcc actaccagct tggtgattga
        5221 ttacagggtt gaggccagcc aagacttctg tgactcctgt ctacagatga tggttcaatc
        5281 cagctccagt tgagacctag tgcaatttcc ctgcagatga acacatagct gtgctttaga
        5341 ctgatgcacc cgtgtcctgt gatggcagga cagggaggct gtgtgctcga gacccatcta
        5401 gacctcgccc tgtttgtgtc tttatttgcc tgcttctgat tctttttctt ttgaataaag
        5461 ttcaaatcat aagtg
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