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.
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 debris at ~10,000rpm in a microcentrifuge for 3 minutes.
5. Remove the entire supernatant (~10ml) and sequence according to the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Protocol (79750)(3).
Sequencing of plasmids in E. coli colonies using the Thermo Sequenase Radiolabeled Terminator Cylce Sequencing Kit.
The rapid preparation of bacterial DNA from colonies described above, in combination with the Thermo Sequenase radiolabeled terminator kit, offers an excellent method to sequence plasmid DNA directly from colonies. Sequence information with virtually no artifacts or background is routinely generated and artifacts associated with template purity or premature termination are not seen at all. Gel compression artifacts, caused by G-C base-pairing of sequencing products, can also be eliminated using the dITP reaction mixture provided with the kit.
The results obtained using these protocols are comparable, or even superior to, those generated using template preparations of high quality and purity. It is important to note that the signal intensity of sequence is related to the size of the colony and the copy number of the plasmid. Therefore, colonies to be sequenced should be at least 2mm in diameter. If the size of the colony is smaller than 2mm or if plasmid is present in low copies, increasing the number of cycles should be beneficial. There is no difference in the quality of sequence generated from fresh or older colonies.
Note: The simple method of colony treatment before sequencing with the Thermo Sequenase radiolabeled terminator cycle sequencing kit was generously offered by Dr. R.S. Ranu at Colorado State University.
1. Life Science News , 20, Amersharn Life Science Inc., pp. 2-4. 2.
2. Fan, J., Ranu, R.S. et al., BioTechniques , 21, (1996), pp. 1132-1137.
3. Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit protocol booklet, 79750, USB Corporation.
Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit, 79750* in USA.
Redivue 33P-labeled terminator pack, AH 9539* from Amersham Pharmacia Biotech
* In the US, the order code is 188403 which includes both 79750 and AH9539.