VDR引物设计出现 灵异事件 忘大侠指教！！
丁香园论坛
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１．小硕我正在做关于维生素Ｄ３受体（ＶＤＲ）多态性实验，准备用ＢｓｍＩ酶切
２．文献上多用Ｍｏｒｒｉｓｏｎ的经典引物：Ｐ１5′CAA ＣCA AGA CTA CAA GTA CCG CGT CAG TGA3′和Ｐ２AAC CAG CGG GAA GAG GTC AAG GG3
３．但是Ｐ２在pubmed上ＢＬＡＳＴ后如下
|74268229|gb|BC103387.1| Bos taurus similar to calcium acti... 34.2 4.1
gi|77735552|ref|NM_001034300.1| Bos taurus similar to calcium... 34.2 4.1
gi|24462304|gb|AC136424.1| Homo sapiens chromosome 5 clone CTD-2 34.2 4.1
gi|24462306|gb|AC008640.6| Homo sapiens chromosome 5 clone CTB-1 34.2 4.1
显然不是ＶＤＲ的
４．我仔细研究后发现此引物有问题，多了一个Ｇ，故改为
Ｐ２AAC CAG CGG AA GAG GTC AAG GG3
经ＢＬＡＳＴ如下：
Sequences producing significant alignments: (Bits) Value
gi|32891816|gb|AY342401.1| Homo sapiens vitamin D (1,25-dihyd... 44.1 0.003
gi|3617739|gb|AC004466.1| Homo sapiens 12 PAC RPCI5-1057I20 (... 44.1 0.003
gi|49654999|emb|CR382137.1| Debaryomyces hansenii chromosome ... 36.2 0.84
gi|50422232|ref|XM_459679.1| Debaryomyces hansenii CBS767, DEHA0 36.2 0.84
gi|62945765|gb|AC160647.1| Gallus gallus BAC clone CH261-14N2... 36.2 0.84
gi|42794112|gb|AC137993.2| Oryza sativa (japonica cultivar-gr... 34.2 3.3
gi|39752700|gb|AC145809.3| Oryza sativa (japonica cultivar-gr... 34.2 3.3
gi|39985517|gb|AE017180.1| Geobacter sulfurreducens PCA, complet 34.2 3.3
gi|21655318|emb|AL137184.15| Human DNA sequence from clone RP... 34.2 3.3
gi|49609491|emb|BX950851.1| Erwinia carotovora subsp. atroseptic 34.2 3.3
gi|58531198|dbj|AP008217.1| Oryza sativa (japonica cultivar-g... 34.2 3.3
gi|17226685|gb|AC083751.6| Genomic sequence for Oryza sativa,... 34.2 3.3
gi|68392477|ref|XM_691473.1| PREDICTED: Danio rerio similar t... 34.2 3.3
gi|77548247|gb|DP000010.1| Oryza sativa (japonica cultivar-gr... 34.2 3.3
gi|22474401|emb|AL354926.17| Human DNA sequence from clone RP... 34.2 3.3
证明没错
５．ＰＣＲ后片段为８００bp与文献吻合左右进一步证实
但问题出来了．这个目的片段不能被ＢｓｍＩ酶切！！！似乎无该酶切位点（ＧＡＡＴＧＣ）！
请各位大侠指正！！！
２．文献上多用Ｍｏｒｒｉｓｏｎ的经典引物：Ｐ１5′CAA ＣCA AGA CTA CAA GTA CCG CGT CAG TGA3′和Ｐ２AAC CAG CGG GAA GAG GTC AAG GG3
３．但是Ｐ２在pubmed上ＢＬＡＳＴ后如下
|74268229|gb|BC103387.1| Bos taurus similar to calcium acti... 34.2 4.1
gi|77735552|ref|NM_001034300.1| Bos taurus similar to calcium... 34.2 4.1
gi|24462304|gb|AC136424.1| Homo sapiens chromosome 5 clone CTD-2 34.2 4.1
gi|24462306|gb|AC008640.6| Homo sapiens chromosome 5 clone CTB-1 34.2 4.1
显然不是ＶＤＲ的
４．我仔细研究后发现此引物有问题，多了一个Ｇ，故改为
Ｐ２AAC CAG CGG AA GAG GTC AAG GG3
经ＢＬＡＳＴ如下：
Sequences producing significant alignments: (Bits) Value
gi|32891816|gb|AY342401.1| Homo sapiens vitamin D (1,25-dihyd... 44.1 0.003
gi|3617739|gb|AC004466.1| Homo sapiens 12 PAC RPCI5-1057I20 (... 44.1 0.003
gi|49654999|emb|CR382137.1| Debaryomyces hansenii chromosome ... 36.2 0.84
gi|50422232|ref|XM_459679.1| Debaryomyces hansenii CBS767, DEHA0 36.2 0.84
gi|62945765|gb|AC160647.1| Gallus gallus BAC clone CH261-14N2... 36.2 0.84
gi|42794112|gb|AC137993.2| Oryza sativa (japonica cultivar-gr... 34.2 3.3
gi|39752700|gb|AC145809.3| Oryza sativa (japonica cultivar-gr... 34.2 3.3
gi|39985517|gb|AE017180.1| Geobacter sulfurreducens PCA, complet 34.2 3.3
gi|21655318|emb|AL137184.15| Human DNA sequence from clone RP... 34.2 3.3
gi|49609491|emb|BX950851.1| Erwinia carotovora subsp. atroseptic 34.2 3.3
gi|58531198|dbj|AP008217.1| Oryza sativa (japonica cultivar-g... 34.2 3.3
gi|17226685|gb|AC083751.6| Genomic sequence for Oryza sativa,... 34.2 3.3
gi|68392477|ref|XM_691473.1| PREDICTED: Danio rerio similar t... 34.2 3.3
gi|77548247|gb|DP000010.1| Oryza sativa (japonica cultivar-gr... 34.2 3.3
gi|22474401|emb|AL354926.17| Human DNA sequence from clone RP... 34.2 3.3
证明没错
５．ＰＣＲ后片段为８００bp与文献吻合左右进一步证实
但问题出来了．这个目的片段不能被ＢｓｍＩ酶切！！！似乎无该酶切位点（ＧＡＡＴＧＣ）！
请各位大侠指正！！！
