有和无甘油的聚丙烯酰胺胶在检测突变时的差别

The Effect of Glycerol in the Polyacrylamide Gel on Detecting Mutation

有文献报道在非变性的聚丙烯酰胺中加入甘油可提高SSCP检测的灵敏度。我们的实验结果建议研究者在进行SSCP筛查未知突变时最好采用不加甘油的非变性的聚丙烯酰胺胶 ,这既省力省钱 ,又灵敏。在判读SSCP胶时 ,千万不要看到在双链带位置有一条比正常迁移率慢的带就判定为插入突变。此时要判定突变的性质 ,最好测序。

It was reported that glycerol in the non-denatured SSCP polyacrylamide gel could increase the sensibility of detecting mutation. We detected the mutation of PKD1 gene in the patients with autosomal dominant polycystic kidney disease.PCR combined with SSCP(single-strained conformation polymorphism),the non-denatured 10% polyacrylamide gel without glycerol or 10% polyacrylamide gel with 5% glycerol and DNA sequencing method were used.Our results showed that four single strand bands were found in the non-denatured polyacrylamide gel without glycerol while two single strand bands were found in the polyacrylamide gel with glycerol in the same patient.Sequence showed there is a deletion of G in one DNA molecular and a G→A substitution in another DNA molecular in the patient with abnormal shift SSCP bands.Therefore, our experiment suggested that non-denatured polyacrylamide gel was better than the polyacrylamide gel withglycerol in detection mutation,and it will save labor and money.It also suggested that one basedeletion can cause a slow double-strand DNA following the normal double strand band,which was caused by the heterogeneous DNA molecule formed between the normal DNA strand and the one base deletion DNA strand with the protruding base.Our results suggest that when judging mutation in SSCP gel,it is not reliable to decide that mutation is inversion according to slow mobility in the gel,and when the characteristic of mutation need to be judged,it must be sequenced.