High performance DNA sequencing, and the detection of mutations and polymorphisms, on the Clipper sequencer

Abstract

The Visible Genetics Clipper sequencer is a new platform for automated DNA sequencing which employs disposable MicroCel cassettes and 50 μm 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 μm thick MicroCel gels. We routinely obtain well‐resolved sequence which can be base‐called with 98.5% accuracy to position ˜450 on an 11 cm gel, and to position ˜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, Δ(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.

Accuracy of the TRUGENEHIV-1Genotyping Kit

“…DNA amplification was performed in the same tube using cycling conditions of 94°C for 30 s, 57°C for 30 s, and 68°C for 2 min for 20 cycles; this was followed by cycling conditions of 94°C for 30 s, 60°C for 30 s, and 68°C for 2.5 min for 17 cycles; and the cycling finished with one cycle of 68°C for 7 min and then a 4°C hold. Unpurified RT-PCR product (5 l) from each specimen was used in each of 16 sequencing reactions, based on the CLIP principle (25), and employing four pairs of primers that sequence the protease reading frame (two pairs) and the beginning and middle of the reverse transcriptase reading frame (one pair each). For each segment, four sequencing reactions were used with four dideoxy terminators (ddATP, ddCTP, ddGTP, and ddTTP), Cy5-labeled forward primers, and Cy5.5-labeled reverse primers to generate CLIP sequencing products over 30 reaction cycles (94°C for 20 s, 56°C for 20 s, 70°C for 1.5 min) followed by a 5-min terminal extension at 70°C.…”

Multicenter Comparative Multimodality Surveillance of Women at Genetic-Familial High Risk for Breast Cancer (HIBCRIT Study): Interim Results

“…Genetic testing for identification of deleterious BRCA1 and BRCA2 mutations included the use of individual or combined procedures, such as direct sequencing (29)(30)(31), protein truncation testing (29), single-strand conformation polymorphism (32), or mutation screening with denaturing chromatography (33).…”

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

“…In 1999, the MicroGene Clipper two-dye automated DNA sequencer (Yager et al 1999) was introduced with a 50-µm ultrathin sequencing gel in a disposable MicroCel cassette. With a Gel Toaster system, the MicroCel cassette can be cast, prepared, and polymerized in <5 min.…”