Info

Full-length fragment Cleaved fragments

■ Figure 9-23 Single-stranded endonucleases cleave mis-paired regions of heteroduplexes (top). The cleaved fragments can be resolved by agarose gel electrophoresis (bottom).

Full-length fragment Cleaved fragments

■ Figure 9-23 Single-stranded endonucleases cleave mis-paired regions of heteroduplexes (top). The cleaved fragments can be resolved by agarose gel electrophoresis (bottom).

ment at the dU sites.101 For example, the sequence to be scanned is amplified in a standard PCR reaction containing a mixture of 0.2 mM dNTPs and 0.015 mM dUTP. One of the primers in the PCR reaction has a fluorescent or radioactive label. With the above ratios of dNTP:dUTP, an average of 1 dU is incorporated into each amplicon. After the PCR reaction, the amplicons are digested with uracil-N-glycosylase and Escherichia coli endonuclease IV to remove the uracils and then cut the sugar phosphate backbone of the DNA. Mutations affecting AT base pairs in the test sequence will be revealed by the incorporation of dU and subsequent fragmentation of the amplicon at the site of dU incorporation. The fragments can then be resolved by gel or capillary electrophoresis (Fig. 9-24). Premixed reagents for this assay are available (BESS T-Scan, Epicentre Technologies).

An extension of this method, the BESS G-Tracker, is designed to interrogate G residues in the test sequence.102 The amplicons, dissolved in a G modification reagent, are subjected to a photoreaction with visible light. A proprietary enzyme mix will then fragment the amplicons at the positions of the modified G residues. Interpretation of the electrophoresis fragment patterns is the same as described above for the T-Scan. BESS is reported to have less optimization requirements than SSCP and ddF.

Normal

Was this article helpful?

0 0

Post a comment