Optimization of protocol for sequencing of difficult templates.
In this paper, we have fine-tuned a DNA sequencing protocol suitable for a wide range of difficult templates. The primary goal was to evaluate a number of parameters--such as various dye terminator mixes in the presence or absence of additives, the amount of DNA or primer, and cycling protocols--about the effectiveness of reading through complex regions. We showed that the modification of a published protocol leads to significant (75%) cost reduction without forfeiting quality of the data. In the recommended protocol, we used betaine as a standard additive, but better results can be obtained when betaine and Reagent A are mixed in an equivalent ratio.
Kieleczawa
Jan
J
Pfizer Research/Global Biotherapeutics Technologies, Cambridge, Massachusetts 02140, USA. j.kieleczawa@verizon.net
Mazaika
Erica
E
D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics*; D017931:DNA Primers; D005456:Fluorescent Dyes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA