Laken S J, Jackson P E, Kinzler K W, Vogelstein B, Strickland P T, Groopman J D, Friesen M D
The Johns Hopkins Oncology Center, Baltimore, MD 21231, USA.
Nat Biotechnol. 1998 Dec;16(13):1352-6. doi: 10.1038/4333.
A method has been developed to produce small DNA fragments from PCR products for analysis of defined DNA variations by mass spectrometry. The genomic region to be analyzed is PCR-amplified with primers containing a sequence for the type IIS restriction endonuclease Bpml. Bpml digestion of the resultant PCR products yields fragments as small as seven bases, which are then analyzed by electrospray ionization mass spectrometry. The approach was validated using seven different variants within the APC tumor suppressor gene, in which a perfect correlation was obtained with DNA sequencing. Both the sense and antisense strands were analyzed independently, and several variants can be analyzed simultaneously. These results provide the basis for a generally applicable and highly accurate method that directly queries the mass of variant DNA sequences.
已开发出一种方法，可从PCR产物中生成小DNA片段，用于通过质谱分析特定的DNA变异。使用含有IIS型限制性内切酶Bpml序列的引物对要分析的基因组区域进行PCR扩增。所得PCR产物经Bpml消化后产生小至七个碱基的片段，然后通过电喷雾电离质谱进行分析。该方法通过APC肿瘤抑制基因内的七个不同变体进行了验证，与DNA测序结果完全相关。对有义链和反义链均进行了独立分析，并且可以同时分析多个变体。这些结果为一种直接查询变异DNA序列质量的通用且高度准确的方法提供了基础。
