甜菜碱提高长距离PCR扩增效率的研究

The improved efficiency of long distance PCR by betaine

目的:利用甜菜碱优化长距离PCR(LD-PCR),提高血友病A(HA)基因倒位检测效率。方法:在LD-PCR反应体系中加入DMSO、甘油及不同浓度的甜菜碱进行LD-PCR扩增,比较扩增结果。用甜菜碱优化LD-PCR检测正常人、不同程度HA患者及携带者的FⅧ基因是否存在FⅧ基因22号内含子(IVS22)倒位。结果:在反应体系中加入甜菜碱可获得更好的LD-PCR扩增结果;甜菜碱优化LD-PCR的最佳终浓度为0.8～1.8mol/L;应用甜菜碱优化LD-PCR检出3例HA患者、2例携带者和1例可疑携带者FⅧ基因有IVS22倒位。结论:一定浓度的甜菜碱可以显著提高LD-PCR的扩增效率,提高检测出HA患者的FⅧ基因IVS22倒位的效率。

Objective To improve the efficiency of long distance polymerase chain reaction （LD-PCR） by using betaine. Methods The PCR enhancers, like dimethyl sulphoxide, glycerol, betaine and betaine of different concentrations were put into the LD-PCR mixture, then the results of amplification were compared. The improved LD- PCR was used to detect if IVS22 was present in the FⅧ gene of the normal controls, hemophilia A patients of different severities and carders. Results Better result of LD-PCR was found in the mixture containing betain, than that with other PCR enhancer or without PCR enhancer. The best concentration of betaine that enhanced LD-PCR was 0.8 - 1.8 mol/L. IVS22 inversion was detected in three hemophilia A patients, two carrier and one possible carrier. Conclusion Betaine of certain concentration can significantly improve the efficiency of LD-PCR amplification and detection of the IVS22 inversion efficiently and correctly.