Sequencing from Large-Insert Clones
Automated sequencing can work with a variety of very large DNA clones such as BAC, Lambda or Cosmid DNA. We've even had some excellent success with direct sequencing from a bacterial genomic template. Here's how to do it:
This page is divided into two sections. The first gives information for the client to help them prepare good quality template DNA and primers. The second section gives information for other Core facilities on how to process BAC DNA for best results. Click here to jump to the latter section.
Section I - template preparation for Core Clients Because large clones like BACs carry with them huge amounts of non-template DNA, the sequencing reaction must be adjusted to help the primer find the correct priming site efficiently. Various other parameters must also be adjusted to compensate for these changes.
Some final comments for Core clients regarding sequencing BACs and other large DNA:
Templates that sequence well will reliably do so. A good BAC preparation will produce ~700 nt of data time and time again, given good primers. However, the same client might prepare a new batch of template with the same protocol, and it may fail miserably time and time again. We do not yet know what makes a BAC prep "good".
Due to these vagaries of DNA preparation, BAC sequencing is simply not as reliable as plasmid sequencing. Our lab is quite experienced with this protocol, yet our success rate is approximately 50%. The difference between success and failure, as always, is with the client's sample. Let's face it - operating a sequencer ain't rocket science. We can't take credit for the successes, but as long as we perform our normal quality control checks, don't be too hasty to blame us for the failures either.
The following is a description of the procedures used in the University of Michigan DNA Sequencing Core for obtaining sequence from large DNA clones, such as BACs, P1s, Lambda and Cosmid clones. Most of our experience is with BAC DNA, so the procedures refer to 'BAC' DNA, but they probably apply equally to the other types of large DNA. See our Table of Template and Primer Concentrations for some info on other large-template types.
To compensate for the large amount of non-template DNA present, BAC sequencing depends on alterations to the reaction recipe and to the cycling conditions that enhance the signal.
Handling the template:
It may be necessary to shear the BAC DNA in order to pipette it. This may, in fact, improve the final sequence.
Reaction Recipe, BigDye V3.1 Chemistry:
Total reaction volume: 23 ul
We used to do modified cycling, but the techs hated to do it, and figured out that most of the time, the regular ABI-recommended cycling worked fine for BACs and bacterial genomes.
Before cycling starts, we heat the samples to 95 degrees for 5 minutes. We then do 25 cycles of:
On more difficult templates, 50 cycles may be necessary; some people use as many as 100 cycles. We rarely go above 25 cycles, and we get excellent results with *good* BAC preps. It has even worked surprisingly well for sequencing directly on bacterial (5MB) genomic DNA!