Fluorescent-based Single-strand Conformation Polymorphism/Heteroduplex Capillary Electrophoretic Mutation Analysis of the P53 Gene

Abstract

Fluorescent-based single-strand conformation polymorphism (F-SSCP) analysis with capillary electrophoresis (CE) is the most common method for the detection of mutation because of its high sensitivity and resolution. In this study, we prepared an inexpensive linear polyacrylamide (LPA), and successfully applied it to CE-SSCP analysis and tandem CE-SSCP/heteroduplex analysis (HA) of the P53 gene on an ABI capillary genetic analyzer. A comparison of the sieving capabilities of a homemade LPA and commercial polydimethylacrylamide (PDMA) demonstrates that the homemade LPA has a higher resolution, a shorter analysis time, and is more suitable for tandem SSCP/HA than commercial PDMA. To show the usefulness, mutations of P53 gene exon 7 -8 in 37 tumor samples were investigated by using homemade LPA. The results indicate that 10 mutations were found in 9 of 37 cases; the majority of P53 mutations were missense mutations, and 70% were located in exon 7, which plays an important role in neoplastic progression in human tumorigenesis.

DNA Diagnostics by Capillary Electrophoresis

“…Peaks of homo- and heteroduplexes provide additional evidence that mutations are present. Thus, a tandem SSCP and duplex analysis improves the sensitivity of mutation detection. ,− Nowadays, certain genes and point mutations in them are so well characterized that they can serve as standards in the development of instrumentation and methodology for CE-SSCP. The p53 tumor suppressor gene is one such example. ,,,,, …”

Simultaneous analysis of microsatellite instability and loss of heterozygosity by capillary electrophoresis with a homemade kit

“…But all above reports made use of an expensive commercial kit (POP-4 matrix and capillary) on the ABI 310 capillary genetic analyzer. In our previous studies, a cheap kit including short-chain linear polyacrylamide matrix (SLPA) and neutral coated capillary was prepared in our laboratory and had been successfully applied to detect gene mutation by SSCP [21][22][23], CDCE [24] and SNaPshot [25]. To our knowledge, there are no reports about the homemade kit for microsatellite analysis on the ABI 310 capillary genetic analyzer, and no systematic study about the effect of urea additive and separation temperature on microsatellite analysis.…”