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

Bjørheim, Jens; Abrahamsen, Torveig Weum; Kristensen, Annette Torgunrud; Gaudernack, Gustav; Ekstrøm, Per Olaf

doi:10.1016/s0027-5107(03)00033-2

Cited by 16 publications

(17 citation statements)

References 23 publications

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“…The technical features of the method allows analysis of DNA variation in several different settings: Single-nucleotide polymorphism (SNP) discovery and identification [9], mutation analysis [10][11][12], quantification of allele copy numbers entering the PCR, e.g., analysis of pooled samples and allelic imbalance [13]. Furthermore, the method has been demonstrated to successfully determine microhaplotypes, i.e., the association of SNP alleles physically spaced over a few hundred bp on a chromosome [14].…”

Section: Introductionmentioning

confidence: 99%

Review of denaturant capillary electrophoresis in DNA variation analysis

Bjørheim

Ekstrøm

2005

Electrophoresis

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Analyses of germline and somatic single-nucleotide DNA variations are important in both population genetics research and clinical practice. Reliable and inexpensive methods that are flexible and designed for automation are required for these analyses. Present day DNA sequencing technology is too expensive for testing all 22-25 000 human genes in populations genetics studies or in scanning large numbers of tumors for novel mutations. Denaturant capillary electrophoresis (DCE) has the potential to meet the need for large-scale analysis of DNA variants. Several different analyses can be performed by DCE, including mutation analysis, single-nucleotide polymorphism (SNP) discovery in individual and pooled samples, detection of allelic imbalance, and determination of microhaplotypes. Here we review the theoretical background of the method, its sensitivity, specificity, detection limit, throughput, and repeatability in the light of current literature in the field.

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Section: Introductionmentioning

confidence: 99%

Review of denaturant capillary electrophoresis in DNA variation analysis

Bjørheim

Ekstrøm

2005

Electrophoresis

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“…According to Santos et al [11], there was a frequency of deletion of 8% in Sri Lankan individuals and Thangaraj et al [12] reported a frequency of 1.85% in the Indian male population. In previous assays for single nucleotide polymorphism/mutation analysis, we have used two fluorescent labels to run standards alongside samples in the same well [13]. As DNA sequencing instruments are capable of measuring up to four fluorescent labels in the same sample, this method may be suitable for running up to four assays in the same well.…”

Section: Discussionmentioning

confidence: 99%

High‐throughput gender determination using automated denaturant gel capillary electrophoresis

Hinselwood¹,

Warren

Ekstrøm³

2005

Electrophoresis

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Sex determination of anonymous samples is a requirement before analysis of DNA variation on X or Y chromosomes. Based on this, we designed a method for screening samples on different DNA capillary sequencing instruments with a sensitivity that is able to quantify sex chromosome abnormalities. The two different amelogenin alleles sited on the X and Y chromosomes were polymerase chain reaction amplified with the same set of primers and separated by denaturant capillary electrophoresis (DCE). Sex chromosome ratios could be reproducibly determined with a relative standard deviation of 8.7%, which is sufficient to distinguish a normal XY karyotype from an XYY karyotype associated with Klinefelter syndrome. Reconstruction experiments demonstrated sensitivity down to a simulated Y:X allelic ratio of 1:127 in all three instruments, enabling the prediction of sex chromosomal aneuploidies. When tested on anonymous pooled and single samples, DCE gave a good prediction of the male to female ratio in pools of 1000 blood donors. In conclusion, DCE is a simple and robust method for sex determination that can be readily performed on commercially available CE systems.

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“…The data is then plotted as midpoint temperature versus the base sequence, creating a melting profile of the fragment 87 . The goal of melting profile analysis of DNA fragments with computer programs prior to DCE is to select and manipulate the target sequence, attach GC-clamp if necessary, so that the region of interest is in a low melting domain adjacent to a sequence with high temperature melting properties 28,53,82,87,[89][90][91] . The introduction of the thermally stable clamp increases the percentage of detectable DNA variants in the low melting domain by DGGE to close to 100% 92,93 .…”

Section: Design Of Dna Amplification Fragments With Appropriate Meltimentioning

confidence: 99%

“…The same method of allele separation was first demonstrated on a commercial capillary DNA sequencing in 2001 73,108 . DCE have been applied to 7 different capillary DNA sequencing instrument, ranging from single capillary up to 384 capillaries 38,56,73,74,78,81,82,84,89,105 . Due to the large variety of instrument and instrument protocols and setting, detailed running descriptions will not be given for each instrument.…”

Section: Denaturant Capillary Electrophoresis (Dce)mentioning

confidence: 99%

“…We have tested two different instruments from one of the leading manufactures of capillary DNA sequencing instruments with respect to their ability to separate alleles by DCE 76,83,89,108 . By use of standard genotyping or DNA sequencing protocols as given for the instruments manuals, except for the temperature setting, we have been able to automatically separate alleles by DCE on the single capillary instrument ABI 310 and the 16 capillary instrument ABI 3100 (figure 11).…”

Section: Instruments From Applied Biosystemsmentioning

confidence: 99%

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Analysis of mutational spectra by denaturing capillary electrophoresis

et al. 2008

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Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75-250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA "clamp" is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human genecommon disease associations and the discovery of origins of point mutations in human development and carcinogenesis.

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

Cited by 16 publications

References 23 publications

Review of denaturant capillary electrophoresis in DNA variation analysis

Review of denaturant capillary electrophoresis in DNA variation analysis

High‐throughput gender determination using automated denaturant gel capillary electrophoresis

Analysis of mutational spectra by denaturing capillary electrophoresis

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