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        如何找到已知引物在mRNA上的的结合位点?

        丁香园论坛

        4096
        请大家帮忙,初次接触PCR,可能问题很菜。

        文献提供的TF的引物为:
        forward, 5-CACTCATCATTGTGGGAGCAGTG-3
        reverse,5-CGCGACGGGGTGTTCTT-3

        请问1:我如何知道这对引物所对应的位点?用何方法或软件?请详细告知?我自己试了试,对不上号:(
          2.这对引物在多篇英文文献中做Real-Time RT-PCR时使用,如果我做半定量RT-PCR,可以使用吗?需要做何修改?

        TF的mRNA在Genbank的详细资料和序列如下:
        1: M26071. Mouse tissue fact...[gi:201924] Links

        LOCUS MUSTF 1821 bp mRNA linear ROD 27-APR-1993
        DEFINITION Mouse tissue factor (mtf) mRNA, complete cds.
        ACCESSION M26071
        VERSION M26071.1 GI:201924
        KEYWORDS membrane-bound glycoprotein; tissue factor.
        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 1821)
        AUTHORS Hartzell,S., Ryder,K., Lanahan,A., Lau,L.F. and Nathan,D.
        TITLE A growth factor-responsive gene of murine BALB/c 3T3 cells encodes
        a protein homologous to human tissue factor
        JOURNAL Mol. Cell. Biol. 9 (6), 2567-2573 (1989)
        MEDLINE 89343974
        PUBMED 2761539
        COMMENT Original source text: Mouse (strain BALB/c) fibroblast 3T3 cell
        line, cDNA to mRNA, and DNA (bp 1-41), clone 3CH 482.
        Draft entry and computer-readable sequence for [1] kindly submitted
        by S.Hartzell, 12-JUL-1989.
        FEATURES Location/Qualifiers
        source 1..1821
        /organism="Mus musculus"
        /mol_type="mRNA"
        /db_xref="taxon:10090"
        mRNA 29..1821
        /product="TFP mRNA"
        CDS 135..1019
        /note="tissue factor protein"
        /codon_start=1
        /protein_id="AAA40414.1"
        /db_xref="GI:201925"
        /translation="MAILVRPRLLAALAPTFLGCLLLQVTAGAGIPEKAFNLTWISTD
        FKTILEWQPKPTNYTYTVQISDRSRNWKNKCFSTTDTECDLTDEIVKDVTWAYEAKVL
        SVPRRNSVHGDGDQLVIHGEEPPFTNAPKFLPYRDTNLGQPVIQQFEQDGRKLNVVVK
        DSLTLVRKNGTFLTLRQVFGKDLGYIITYRKGSSTGKKTNITNTNEFSIDVEEGVSYC
        FFVQAMIFSRKTNQNSPGSSTVCTEQWKSFLGETLIIVGAVVLLATIFIILLSISLCK
        RRKNRAGQKGKNTPSRLA"
        ORIGIN Unreported.
        1 tttataacgc accccgcgcg accccggcag cctgggtaca gccggtaccc atcactcgct
        61 ccctccgatc gctcctgtag cgtagccaac gcgccgccgc tgaagccccg agacctcgcc
        121 tccagccctt ggacatggcg atcctcgtgc gcccgcgcct cctagcggct ctcgcgccca
        181 cgtttctcgg ctgcctcctc ctccaggtga ccgcgggtgc aggcattcca gagaaagcgt
        241 ttaatttaac ttggatatca actgatttca agacaatttt ggagtggcaa cccaaaccca
        301 ccaactatac ctacactgta cagataagtg atcgatctag aaactggaaa aacaagtgct
        361 tctcgaccac agacaccgag tgcgacctca cagacgagat cgtgaaggat gtgacctggg
        421 cctatgaagc aaaggtcctc tctgtcccac ggaggaactc agttcatgga gacggagacc
        481 aacttgtgat tcatggggag gagccgccat ttacaaacgc cccaaagttt ttaccttacc
        541 gagacacaaa ccttggacag ccagtaattc agcagtttga acaagatggt agaaaactga
        601 acgtggttgt aaaagactca cttacattag tcagaaagaa tggtacattc ctcaccctgc
        661 ggcaagtctt tggcaaggac ttgggttata taattactta tcggaaaggc tcaagcacgg
        721 gaaagaaaac aaacattaca aacaccaatg aattctcgat tgatgtggaa gaaggagtaa
        781 gctactgctt ttttgtacaa gctatgattt tctccaggaa aactaaccaa aatagcccag
        841 gaagcagtac agtgtgcacc gagcaatgga agagtttcct gggagaaaca ctcatcattg
        901 tgggagcagt ggtgctcctg gccaccatct ttatcatcct cctgtccata tctctgtgca
        961 agcgcagaaa gaaccgagcg ggacagaaag ggaagaacac cccgtcgcgc ttggcataga
        1021 ggaaaggctg aagcgctaac gctcacactg cctgcacggc actgttgcgg agagctctga
        1081 tgggaactgt gcaacatgga gcgtggagcc tgccgatcct agctcagaga ggctgccttc
        1141 atggcctgtt actccagcta acgctttgat tccaacacta gcatttgtca cgttaggacg
        1201 aactgaaacg gtacaaactg gttaacacta cagcgccttt tgcacaaatg ctttagattg
        1261 tatggtctac actcaggaag acactaggtc acccaggcaa agccagtgga cagatgcctt
        1321 tcatataacc tgggtgggct tttggaaaat ctttgagaag ttgatttcat aggctgtaga
        1381 acagtaaagt gggaactggg cggacttttc taacagtcgt acttttataa agcggtattt
        1441 gggtgttttt tccctcgaat aggtactttt ggaagttcaa agcaagtggc aaactttcat
        1501 ataaacatgt taaatgcagg atatttctgc ttggggcatc tttgtgattt gtactttcct
        1561 acaatttagc actttaactg acaatgatgg ggtttaaaca tttgacagcc aactctattt
        1621 ttatacgact actatacaaa gaaactacat atagttttat gatttaatgg ttaacattgt
        1681 atatatttac ataaaattta ggtacttaga aatgtttaaa ggttttgtat atgggatttt
        1741 ctatttatat agcttctatt tgtatatttt gagataattt atttaatata cttttatata
        1801 aataaaggtg attgggaatt g
        //
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