Separation principles of cycling temperature capillary electrophoresis

Abstract

High throughput means to detect and quantify low-frequency mutations (<10(-2) ) in the DNA-coding sequences of human tissues and pathological lesions are required to discover the kinds, numbers, and rates of genetic mutations that (i) confer inherited risk for disease or (ii) arise in somatic tissues as events required for clonal diseases such as cancers and atherosclerotic plaque.While throughput of linear DNA sequencing methods has increased dramatically, such methods are limited by high error rates (>10(-3) ) rendering them unsuitable for the detection of low-frequency risk-conferring mutations among the many neutral mutations carried in the general population or formed in tissue growth and development. In contrast, constant denaturing capillary electrophoresis (CDCE), coupled with high-fidelity PCR, achieved a point mutation detection limit of <10(-5) in exon-sized sequences from human tissue or pooled blood samples. However, increasing CDCE throughput proved difficult due to the need for precise temperature control and the time-consuming optimization steps for each DNA sequence probed. Both of these problems have been solved by the method of cycling temperature capillary electrophoresis (CTCE). The data presented here provide a deeper understanding of the separation principles involved in CTCE and address several elements of a previously presented two-state transport model.

Tp53/p53 status in keratoacanthomas

“…This is clearly supported by the fact that p53 positivity is higher in KAs with mutations. Tp53 gene mutations were detected by cycling temperature capillary electrophoresis which is a simple, sensitive and robust method to detect the presence of mutation in the hot spot regions of exon 5–8 of the Tp53 gene, which is located on chromosome arm 17p. As we did not sequence the cases positive for Tp53 mutation, we do not know the types of mutations in our material, only in which exon they are located.…”

“…Mutation analysis for Tp53 was performed by cycling temperature capillary electrophoresis according to Ekstrøm et al This procedure detected the presence of gene mutations in the hot spot regions of exons 5–8. The method was chosen because it could answer our question about the frequency of Tp53 mutations in KAs in a reliable way with high detection sensitivity (0.1%, compared with about 30% for Sanger sequencing), and because it is only moderately time consuming.…”

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis

“…CTCE analysis was performed for the selected fragments as previously described [28]. In brief; a 96-capillary DNA analyzer (MegaBACE 1000) was used to analyze 6-carboxyfluorescein labeled PCR products.…”

Ten modifiers of BRCA1 penetrance validated in a Norwegian series

“…Default Primer3 ( http://bioinfo.ut.ee/primer3/ , last accession date 03062014) parameters were applied when designing primers used to amplify fragments around each DNA variant, identified by the NCBI SNP reference numbers (rs) [ 16 ]. A 42-bp artificial high melting domain, labeled with 6-FAM, was incorporated at one end of the amplified target using a set of three primers in the PCR setup [ 17 ].…”