Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.
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 BsaJ I and EcoR 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.
Shi
Liang
L
Torrey Mesa Research Institute, San Diego, CA 92121, USA.
Khandurina
Julia
J
Ronai
Zsolt
Z
Li
Bi-Yu
BY
Kwan
Wai King
WK
Wang
Xun
X
Guttman
András
A
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D016172:DNA Fingerprinting; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D019075:Electrophoresis, Capillary / Q000379:methods*; D020224:Expressed Sequence Tags; D005456:Fluorescent Dyes; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D004247:DNA; D015246:Deoxyribonuclease EcoRI; C047012:endodeoxyribonuclease SecI; D015252:Deoxyribonucleases, Type II Site-Specific