Rapid screening for Tp53 mutations by temperature gradient gel electrophoresis: a comparison with SSCP analysis.
Rapid screening for Tp53 mutations by temperature gradient gel electrophoresis: a comparison with SSCP analysis.
复制标题
通过温度梯度凝胶电泳快速筛查 Tp53 突变：与 SSCP 分析的比较。
DOI：
10.1093/hmg/2.12.2155
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发表时间：
1993
影响因子：
3.5
通讯作者：
T. Löning
中科院分区：
文献类型：
--
作者：
R. Scholz;K. Milde‐Langosch;R. Jung;H. Schlechte;H. Kabisch;C. Wagener;T. Löning
Figure 2. TGGE analysis of 20 cases of colon carcinoma samples,—negative control= without DNA, B= BXPC 3, C= CEM, A= A 431, and B/C= BXPC3 and CEM mixed and denaturated and renatured. mutations are marked by asterisks. TGGE was performed under following conditions: exon 5: run-in time (20 Q: 90 min, temperature gradient: 45 C-75 C, running time: 4 h, PCR primer forward (GC40) nt 13040-13059, reverse: nt 13281-13261; exon 6: run-in time (20 Q: 100 min, gradient: 35 C-68 C, running time 2 h 50 min, primer forward (GC40) nt 13261-13281, reverse: nt 13447-13428; exon 7: run-in time: 30 min, gradient: 40 C-75 C, running time 2 h 30 min, primer: forward (GC40) nt 13992-14013, reverse: nt 14125-14105; exon 8: run-in time: 3h, gradient: 4OC-78 C, running time: 3 h, primer: forward nt 14438-14459, reverse (GC40) nt 14612-14591. Primer sequences according to PC Lamb and L. Crawford [20] were obtained from Dr VC Sheffield, Iowa City. For better detection of single base changes, one of the primers had an attached GC-datnp (=-GC 40;[10]). Amplified fragments were purified by phenol/chloroform extraction and ethanol precipitation and redissolved in 30 jd TGGE running buffer (20 mM MOPS, 1 mM EDTA, pH 8.0) prior to TGGE.Among the different screening techniques for the detection of subtle mutations SSCP analysis is die most widely used mediod and involves separation of single DNA strands on nondenaturing polyacrylamide gels. Mutations are identified by mobility shifts due to a change in secondary structure. Recently Sakar et al. showed, that the sensitivity of this method is highly dependant on the length of the analysed fragment [21]. For optimal results, parallel gels should be run under different conditions (with and without glycerol, different temperatures, different polyacrylamide concentrations).
