MALDI-TOF-MS结合引物延伸反应检测K-ras基因型方法的建立

Establishment and Application of Research Model for Screening the K-ras Alleles by MALDI-TOF-MS Coupled with Primer Extension Reaction Method

目的:建立MALDI-TOF-MS结合引物延伸的方法检测K-ras基因12位密码子7种基因型的研究模型并探讨其应用价值。方法:以自行构建的K-ras质粒DNA为模板,经目的片断扩增,去磷酸化,再用两条未经修饰的普通引物,在dATP、ddTTP、ddCTP和ddGTP混和物存在的体系中进行引物延伸反应,最后用MALDI-TOF-MS检测引物延伸产物质荷比(m/z),得到质谱图,根据质谱图中各个峰的峰值进行基因分型研究。结果:用MALDI-TOF-MS结合引物延伸的方法,可以准确区分7种不同基因型。该方法可同时对384份样品进行检测。结论:MALDI-TOF-MS结合引物延伸的方法准确度高,反应体系稳定并具有快速、高通量、自动化的特点,可能成为肿瘤早期诊断的检测方法。

Objective: To establish a research model for screening seven different genotypes of codon 12 in K-ras alleles and to discuss its clinical application. Methods: In this assay, the man-made plasmid DNAs were used as the templates. First specific DNA fragments containing the target site(s) were amplified from the plasmid DNA, then through sample dephosphorylation, and two ordinary oligodeoxyribonucleotide primers without any modification were used, followed by the primer extension reaction in the presence of dATP, ddTTP, ddCTP and ddGTP mixture, the products were then analyzed using MALDI-TOF mass spectrometry by detecting the m/z ratio. According to the peak value in the mass spectrogram the genotypes were analyzed. Results: The unambiguous result could be achieved through MALDI-TOF-MS coupled with primer extension method and 384 samples could be analyzed simultaneously. Conclusions: MALDI-TOF-MS coupled with primer extension method is accurate and stable, and the reaction system takes the advantage of automation and high-throughput. It is likely to be an alternative approach in early diagnosis and cancer screening in future.