Simultaneous detection of size and sequence polymorphisms in the transcribed trinucleotide repeat D2S196E (EST00493)

Abstract

Long expansions of transcribed trinucleotide microsatellites have been etiologically associated with some neurological diseases. The investigation of such novel polymorphisms has thus become a subject of great interest. We searched the expressed sequence tag databank for reiterated trinucleotides and selected EST00493 (D2S196E) with 14 tandem ACA triplets as a potentially polymorphic locus. Size variation was readily detected, with four common alleles containing 12-15 repeats. In addition, we observed distinct heteroduplexes in amplifications from individuals with identical ACA genotypes. Sequencing of their polymerase chain reaction (PCR) products revealed a G-->A transition immediately preceding the trinucleotide repeats, hence defining 8 distinct haplotypes and 36 possible genotypes. Indeed, mutation detection enhancement gel electrophoresis of mixed PCR products from cloned haplotypes revealed 24 distinct heteroduplex patterns for the six possible trinucleotide heterozygotes. The observation of heteroduplex patterns in non-denaturing polyacrylamide gel electrophoresis (instead of the more commonly used denaturing gels) can thus be utilized to increase the informativeness of microsatellite polymorphisms by unraveling otherwise cryptic sequence variation. The D2S196E polymorphism has proved useful for demonstrating microsatellite instability and loss of heterozygosity in colorectal tumors.

Fully mutated and gray-zoneFRAXA alleles in Brazilian mentally retarded boys

“…In another patient who had a full mutation by the screening test and mosaicism by Southern blot analysis, a normal range allele was amplified using PCR in a second DNA extraction of the same blood sample. All DNA samples from possible mosaic cases were tested for identity with other polymorphic microsatellites D12S67, D13S308E, and D2S196E [Roewer et al, 1992;Haddad and Pena, 1993;Haddad et al, 1997], and no other discrepancy was detected (data not shown).…”

DNA Based Techniques for Studying Genetic Diversity

“…Searches of EST databases for microsatellite containing sequences have been useful for a number of species including humans (Haddad et al, 1997), catfish (Serapion et al, 2004), rice (Cho et al, 2000) and barley (Thiel et al, 2003). In Rainbow trout, cDNAs and expressed sequence tags (ESTs) available in public databases offer an in silico approach to marker development at virtually no cost.…”

Utilization of microsatellites for the analysis of genomic alterations in colorectal cancers in Brazil

“…Most studies of microsatellite instability have been based on the study of (CA) n repeats, which are abundant and sensitive, but cumbersome to type because alleles can only be easily resolved on sequencing gels and generally require isotopic labeling for visualization of the PCR products. In contrast, as we have shown elsewhere, microsatellites with repeats of more than 3 bp can be much more simply typed in short non-denaturing gels with non-isotopic silver staining (17)(18)(19). We report here a very simple methodology based on a set of tri-, tetra-and pentanucleotide repeat microsatellites which allows the simultaneous study of microsatellite instability and loss of heterozygosity and thus helps to allocate a given colorectal tumor to the classic or mutator pathway.…”