High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer.
The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 microm thick polyacrylamide gels. Two DNA ladders can be analyzed simultaneously in each of 16 lanes on a gel, after labeling with far-red absorbing dyes such as Cy5 and Cy5.5. This allows a simultaneous bidirectional sequencing of four templates. We have evaluated the Clipper sequencer, by cycle-sequencing of an M13 single-stranded DNA standard, and by coupled amplification and sequencing (CLIP) of reverse-transcribed human immunodeficiency virus (HIV-1) RNA standards and clinical patient samples. (i) Limitations of instrument. We have examined basic instrument parameters such as detector stability, background, digital sampling rate, and gain. With proper usage, the optical and electronic subsystems of the Clipper sequencer do not limit the data collection or sequence-determination processes. (ii) Limitations of gel performance. We have also examined the physics of DNA band separation on 50 microm thick MicroCel gels. We routinely obtain well-resolved sequence which can be base-called with 98.5% accuracy to position approximately 450 on an 11 cm gel, and to position approximately 900 on a 25 cm gel. Resolution on 5 and 11 cm gels ultimately is limited by a sharp decrease in spacing between adjacent bands, in the biased reptation separation regime. Fick's (thermal) diffusion appears to be of minor importance on 6 cm or 11 cm gels, but becomes an additional resolution-limiting factor on 25 cm gels. (iii) Limitations of enzymology. Template quality, primer nesting, choice of DNA polymerase, and choice between dye primers and dye terminators are key determinants of the ability to detect mutations and polymorphisms on the Clipper sequencer, as on other DNA sequencers. When CLIP is used with dye-labeled primers and a DNA polymerase of the F667Y, delta(5'--> 3' exo) class, we can routinely detect single-nucleotide mutations and polymorphisms over the 0.35-0.65 heterozygosity range. We present an example of detecting therapeutically relevant mutations in a clinical HIV-1 RNA isolate.
Yager
T D
TD
Visible Genetics, Toronto, ON, Canada. tom@visgen.com
Baron
L
L
Batra
R
R
Bouevitch
A
A
Chan
D
D
Chan
K
K
Darasch
S
S
Gilchrist
R
R
Izmailov
A
A
Lacroix
J M
JM
Marchelleta
K
K
Renfrew
J
J
Renfrew
J
J
Rushlow
D
D
Steinbach
E
E
Ton
C
C
Waterhouse
P
P
Zaleski
H
H
Dunn
J M
JM
Stevens
J
J
D017104:Bacteriophage M13 / Q000235:genetics*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D005782:Gels; D005838:Genotype; D015497:HIV-1 / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes; D005782:Gels; D012367:RNA, Viral