如何找到已知引物在mRNA上的的结合位点？
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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
//
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
//
