Biotechnology
| Poster #511 | |
| » | Abstract |
| » | Methods |
| » | Resolution, Read Length, Accuracy |
| » | Human BRCA1 Exon 11 Analysis |
| » | Summary |
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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
Methods
During amplification a limiting amount of deoxyuridine is randomly incorporated in place of deoxythymidine in the PCR product. The primers are labeled with either IRD700 or IRD800 for direct detection.
The PCR product is incubated with the BESS T-Scan Excision Enzyme Mix that contains Uracil glycolyase that removes the uracil bases from the product, creating abasic sites that are immediately cleaved by Exonucleas IV. This process generates a pattern of DNA fragments. (See Figure 1 below.)
A DNA ladder virtually identical to a "T" lane sequencing ladder is produced, representing the pattern of dUTP incorporation and cleavage.
Changes in DNA sequence are detected by changes in banding patterns as compared to a normal or control pattern
Mutations can be detected in both strands from a single reaction using two primers labeled with different dyes as shown in BRCA1 example.
