John M. S. Bartlett.pdf - Bio-Nica.info

Sequencing Difficult Templates 401
58
Technical Notes for Sequencing Difficult Templates
David Stirling
1. Introduction
There are a number of template types that are generally recognized as being difficult
to sequence. These can include sequences with a high guanine–cytosine (G/C) content,
sequences that are very rich in adenine/thymine (A/T), sequences with a marked
secondary structure, or large regions of homopolymer. There is no one solution to
these difficulties; however, there are a number of approaches that can be used to improve
the quality of sequencing data obtained from each type of problem sequence.
2. High G/C Content
Sequences with high G/C content have higher melting temperatures, and the
incorporation of the dye-labeled terminators is also less efficient. The addition of
DMSO to a final concentration (v/v) of 5% or betaine (final concentration of 1 M ) to
cycle sequencing reactions can greatly improve results. Changing the cycling conditions
to use a higher melting temperature can help, as can the use of altered sequencing
chemistry. Applied Biosystems produce a dGTP Big Dye kit where dITP normally
used in big dye chemistry is replaced with dGTP. Adding more Taq and dNTPs can
also help.
3. A/T-Rich Sequences
A / T-rich sequences are generally not as difficult as G/C-rich templates. A /T-rich
primers will have low melting temperatures and so may need to be longer (24–26 bases)
to increase the melting temperature closer to 55°C. Dye primer sequencing is generally
more even through A /T regions.
4. Secondary Structure
Sudden loss of sequencing data generally indicates a problem with secondary
structure. Where there are runs of Gs followed by runs of Cs, for instance, hairpin loops
can form, impeding the progress of the polymerase and resulting in a stop in the data.
Any of the approaches suggested for high G/C content sequences can be used.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
401