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Micropreparative capillary gel electrophoresis of DNA: Rapid expressed sequence tag library construction
Author(s) -
Shi Liang,
Khandurina Julia,
Ronai Zsolt,
Li BiYu,
Kwan Wai King,
Wang Xun,
Guttman András
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390035
Subject(s) - capillary electrophoresis , gel electrophoresis , microbiology and biotechnology , biology , polymerase chain reaction , complementary dna , dna , chromatography , chemistry , genetics , gene
A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment‐pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by Bsa J I and Eco R I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high‐resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer‐controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96‐well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments ( i.e. , single‐base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR‐amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.