Biotechnology
| Poster #511 | |
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| » | Resolution, Read Length, Accuracy |
| » | Human BRCA1 Exon 11 Analysis |
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Detection of Human DNA Mutations using Base Excision Sequence Scanning (BESS) and the LI-COR IR2 Automated DNA Sequencer
Harry Osterman and Gi Y. Jang - LI-COR Inc., Biotechnology Division, Lincoln, NE 68504
Abstract
Single nucleotide polymorphism (SNP) analysis will be quite useful in the study of complex genomes. However, it will first be necessary to locate large numbers of SNP's before routine SNP screening can take place. Identification of SNP loci involves the examination of large numbers of samples for given loci of a genome, primarily using electrophoresis-based mutation analysis.
Base Excision Sequence Scanning (BESS) (Epicentre Technologies, Madison, WI) offers a fast and reliable method for detection of DNA mutations as well as SNP's. In a BESS "T-scan™", PCR is performed using one labeled primer (or two differentially-labeled primers) in the presence of dUTP. After PCR the DNA is enzymatically cleaved at the site of dU incorporation, producing a set of nested DNA fragments that are then separated and detected using high resolution gel electrophoresis. A pattern similar to "T" lane sequencing is produced, allowing direct analysis of different DNA samples for mutational differences, including the detection of SNP's. The BESS fragments run one base quicker than Sanger-based sequencing fragments since BESS fragments are created by dU removal, whereas Sanger-based fragments are terminated by ddT addition.
We report the use of the BESS technique in combination with the LI-COR IR2 Automated DNA Sequencer. The image presentation format available with this sequencer, in combination with up to 96 lanes, provides a useful and quick method for visualizing mutation differences among samples. Additionally, the long read capability of this instrument allows for higher throughput of SNP detection. Polymorphic bands of the mitochondrial control region from 12 different individuals were detected and confirmed by comparison to DNA sequencing results. A G > T transversion in the BRCA1 gene (codon 1250) was easily detected using the BESS technique as well as the ability to detect mutations in heterozygotes by the analysis of band patterns.
