( Introduction In ...)A simple method to sequence from bacterial colonies using [a-33P] radiolabeled ddNTPs and Thermo Sequenase

Introduction

In traditional DNA sequencing systems, sequencing products are radioactively labeled using either endlabeled primers or by the incorporation of radiolabeled dNTPs during primer extension. Successful sequencing results produced by these two labeling methods are highly dependent on template quality. DNA containing nicks, breaks, impurities or high secondary structure can cause DNA polymerases to dissociate from the primertemplate complex before specific ddNTP incorporation occurs. This non-specific termination produces bands across all four lanes (BAFLs) or background artifacts, usually making sequence interpretation difficult. These artifacts can be avoided by the use of [(a-33P] labeled ddNTPs. Labeled terminators prevent the visualization of sequencing artifacts because only specifically-terminated primer extension products are labeled.

The Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit(l) employs a one-step cycle sequencing method that allows the use of as little as 20 fmoles of template. Such high sensitivity allows sequencing of plasmid DNA directly from bacterial colonies with minimal purification. This is extremely useful when rapid screening of many different clones is required. The use of Thermo Sequenase and 33P-labeled ddNTPs, combined with the colony preparation outlined below(2), allows for direct colony sequencing with superior results.

Method

1. Select an appropriate sized colony (~2mm in diameter) on the agar plate and aseptically transfer the entire colony to 10ml of delonized water in a sterile 0.5ml tube.

2. Carefully swirl the colony in water with a sterile 200ml pipet tip. Pipet up and down several times until the colony is fully resuspended.

3. Heat the cell suspension at 95C for 5 minutes, then transfer the tube to an ice bath for 5 minutes.

4. Spin down the cell
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