Jens Bjørheim

Mutation analyses of KRAS exon 1 comparing three different techniques : Temporal temperature gradient electrophoresis, constant denaturant capillary electrophoresis and allele specific polymerase chain reaction

Mutations in the KRAS gene is a key event in the carcinogenesis of many human cancers and may serve as a diagnostic marker and a target for therapeutic intervention. In this study we have applied three different techniques for mutation detection of KRAS exon 1 mutations: Allele specific polymerase chain reaction (AS-PCR), temporal temperature gradient electrophoresis (TTGE) and constant denaturant capillary electrophoresis (CDCE). Samples from 191 sporadic colon carcinomas were analyzed. AS-PCR were performed with oligonucleotides specific for know mutations in codon 12 and 13 of the KRAS gene. In TTGE analyses, linear ramping of the temperature were performed during electrophoresis in a constant denaturant gel. CDCE analyses were performed using fluorescin labeled PCR-products. Separation was achieved under constant denaturing conditions using high temperature in a gel-filled capillary followed by laser detection. A mutated KRAS gene was found in 42/191 (22.0%) of the samples using AS-PCR, in 62/191 (32.5%) using TTGE and in 66/191 (34.6%) of the samples using CDCE. In the TTGE and CDCE analyses the sequence of the mutant were determined by comparing the electrophoretic pattern to that of known mutations or by mixing the sample with known mutations prior to reanalysis. In a titration experiment mixing mutant and wild-type alleles prior to PCR, the sensitivity for mutation detection was shown to be 10(-2) for TTGE and under optimized conditions 10(-3) for CDCE.

Population screening of single-nucleotide polymorphisms exemplified by analysis of 8000 alleles.

The authors describe a method in which the population frequency of single-nucleotide polymorphisms (SNPs) can be efficiently detected and their allele frequencies accurately measured. Selected SNPs in TNF , IL-4 , and CTLA-4 were used to demonstrate the method. Blood from 4000 individuals was pooled, DNA was extracted, and target sequences were PCR amplified and analyzed by denaturant capillary electrophoresis. Alleles were separated into peaks based on melting properties of the double DNA helix. Frequencies of the different alleles were determined by calculating the area under the peaks. Allele frequencies and Hardy-Weinberg equilibrium estimated from the pooled data were verified by analyzing 7.5% of the samples randomly selected from the blood donor series. The method herein is equally suitable for single-samples and/or pooled-samples analysis of SNPs, in which sample treatment is kept to a minimum. The potential throughput of the method is beyond obtainable numbers of samples.

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.

DNA variants in the ATM gene are not associated with sporadic rectal cancer in a Norwegian population-based study

Background and aimsA large number of DNA single-nucleotide polymorphisms (SNPs) have been discovered following the Human Genome Project. Several projects have been launched to find associations between SNPs and various disease cohorts. This study examined the possible association between the reported SNPs and sporadic rectal cancer. It has been proposed that SNPs in the ataxi-telangiectasia mutated (ATM) gene modulate the penetrance of some cancers. The investigated target sequence harbors three polymorphisms (IVS38-8 T/C in intron 38, 5557 G/A and 5558 A/T in exon 39), resulting in eight possible microhaplotypes at the DNA level. Furthermore, the two exonic SNPs are sited next to each other, allowing four possible amino acids in the same codon.MethodsWe report on a new method analyzing SNPs and microhaplotypes based on theoretical thermodynamics and migration of variant fragments by cycling temperature capillary electrophoresis. Fluorophore-labeled PCR products were analyzed without any post-PCR steps on a standard 96 capillary-sequencing instrument under denaturing conditions.ResultsMore than 7000 alleles were microhaplotyped based on peak migration patterns of individual samples and sequencing results. The ATM polymorphisms and microhaplotypes examined did not significantly differ between sporadic rectal cancer and normal population.ConclusionNo associations were found between the IVS38-8 T/C, 5557 G/A and 5558 A/T polymorphisms and microhaplotypes in the ATM gene with respect to sporadic rectal cancer.

Melting gel techniques in single nucleotide polymorphism and mutation detection: From theory to automation

During the last 20 years several applications and variations of the melting gel techniques have been used for mutation and single nucleotide polymorphism analysis. All melting gel techniques are based on the same theoretical platform and are used by large groups in the research community. This review reports on current methods and applications in the field of melting gel techniques.