PCR template preparation for capillary DNA sequencing.
Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.
Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Brazil.
W A
WA
Silva
M C
MC
Costa
V
V
Valente
J F
JF
Sousa
M L
ML
Paçó-Larson
E M
EM
Espreafico
S S
SS
Camargo
E
E
Monteiro
A J
AJ
Holanda
M A
MA
Zago
A J
AJ
Simpson
E
E
Dias Neto
D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA