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请大家给我看看这对引物是不是有问题

丁香园论坛

1085
我的课题涉及到CYP1A1的多态,从文献上(cancer res等级别的文章,而且重复的人挺多)查到它的一个MspI多态位点引物为5’-CAG TGA AGA GGT GTA GCC GCT-3'和5’-TAG GAG TCT TGT CTC ATG CCT-3’,但我有扩增出了其他引物的条件都没有能扩增出这对引物的目标带,做梯度从48到65度都没有带出来。下面是所查的该引物的Blast的资料和该基因这个位点的基因序列资料,请高手帮忙分析一下到底这对引物可不可以扩出来
Blast资料:

>gi|219566|dbj|D12525.1|HUMCYP1A12 Homo sapiens gene for cytochrome P450IA1, 3'flanking region
Length = 650

Score = 44.1 bits (22), Expect = 0.013
Identities = 22/22 (100%)
Strand = Plus / Minus


Query: 21 ttaggagtcttgtctcatgcct 42
||||||||||||||||||||||
Sbjct: 606 ttaggagtcttgtctcatgcct 585

Score = 42.1 bits (21), Expect = 0.050
Identities = 21/21 (100%)
Strand = Plus / Plus


Query: 1 cagtgaagaggtgtagccgct 21
|||||||||||||||||||||
Sbjct: 266 cagtgaagaggtgtagccgct 286

>gi|13430063|gb|AF253322.1|AF253322 Homo sapiens cytochrome P450 (CYP1A1) and cytochrome P450 (CYP1A2)
genes, complete cds
Length = 43064

Score = 44.1 bits (22), Expect = 0.013
Identities = 22/22 (100%)
Strand = Plus / Plus


Query: 21 ttaggagtcttgtctcatgcct 42
||||||||||||||||||||||
Sbjct: 4285 ttaggagtcttgtctcatgcct 4306

Score = 42.1 bits (21), Expect = 0.050
Identities = 21/21 (100%)
Strand = Plus / Minus


Query: 1 cagtgaagaggtgtagccgct 21
|||||||||||||||||||||
Sbjct: 4628 cagtgaagaggtgtagccgct 4608

基因序列

1: D12525. Homo sapiens gene...[gi:219566] Links

LOCUS HUMCYP1A12 650 bp DNA linear PRI 13-FEB-2003
DEFINITION Homo sapiens gene for cytochrome P450IA1, 3'flanking region.
ACCESSION D12525 D01198
VERSION D12525.1 GI:219566
KEYWORDS Benzo(a)pyrene metabolism.
SOURCE Homo sapiens (human)
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.
REFERENCE 1
AUTHORS Kawajiri,K., Watanabe,J., Gotoh,O., Tagashira,Y., Sogawa,K. and
Fujii-Kuriyama,Y.
TITLE Structure and drug inducibility of the human cytochrome P-450c gene
JOURNAL Eur. J. Biochem. 159 (2), 219-225 (1986)
MEDLINE 87004629
PUBMED 3019683
REFERENCE 2
AUTHORS Kubota,M., Sogawa,K., Kaizu,Y., Sawaya,T., Watanabe,J.,
Kawajiri,K., Gotoh,O. and Fujii-Kuriyama,Y.
TITLE Xenobiotic responsive element in the 5'-upstream region of the
human P-450c gene
JOURNAL J. Biochem. 110 (2), 232-236 (1991)
MEDLINE 92105053
PUBMED 1761516
REFERENCE 3 (bases 1 to 650)
AUTHORS Hayashi,S., Watanabe,J., Nakachi,K. and Kawajiri,K.
TITLE Genetic linkage of lung cancer-associated MspI polymorphisms with
amino acid replacement in the heme binding region of the human
cytochrome P450IA1 gene
JOURNAL J. Biochem. 110 (3), 407-411 (1991)
MEDLINE 92121159
PUBMED 1722803
REFERENCE 4 (bases 1 to 650)
AUTHORS Hayashi,S.
TITLE Direct Submission
JOURNAL Submitted (04-OCT-1991) Shin-ichi Hayashi, Saitama Cancer Center
Research Institute, Department of Biochemistry; 818 Komuro,
Inamachi, Saitama 362, Japan (Tel:81-48-722-1111(ex.257),
Fax:81-48-722-1739)
FEATURES Location/Qualifiers
source 1..650
/organism="Homo sapiens"
/mol_type="genomic DNA"
/db_xref="taxon:9606"
/chromosome="15"
/map="15q22-qter"
/clone="phP450mc-2"
/cell_type="lymphocyte"
/tissue_type="blood"
/note="germline"
polyA_signal 132..137
/note="cytochrome P450IA1 (CYP1A1)"
variation 400
/note="Base substitution has occurred at this position in
genotype C
MspI polymorphism
cancer susceptibility associated"
/replace="c"
ORIGIN
1 acaatccttc tattctagcc tgcattgagc ttgcatgctt gcataagagc ttaagaaacc
61 attgatttaa tgtaataggg aaaattctaa cccaggtatc caaaaatgtg taagaacaac
121 tacctgagct aaataaagat attgttcaga aaatcctata ggtggagatt ttttgaatca
181 taaatgattc atcactcgtc taaatactca ccctgaaccc cattctgtgt tgggttttac
241 tgtagggagg aagaagagga ggtagcagtg aagaggtgta gccgctgcac ttaagcagtc
301 tgtttgaggg acaagactct attttttgag acagggtccc caggtcatcc aggctggagt
361 gcactggtac cattttgttt cactgtaacc tccacctcct gggctcacac gattctccca
421 cctcagcctc tgagtagttg gggccgccag acgccaccac agcttttttt tttttttttt
481 tttttttttg tagagatggg gtttcaccat gttgcccagg ctggtctcaa actcctgagc
541 tcaagtgatc cacctgcctc agcctcccaa agtgctggga ttacaggcat gagacaagac
601 tcctaatcac tgtgctgtct tagcgccctc tctaacttat cacaaattga
//
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