TY  - BOOK
AU  - Ranade,S.S.
AU  - Chung,C.B.
AU  - Zon,G.
AU  - Boyd,B.L.
TI  - Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection
KW  - DNA
KW  - FRAGMENT LIBRARY
KW  - BISULFITE
KW  - POLYACRYLAMIDE GEL ELECTROPHORESIS
KW  - SINGLE-MOLECULE PCR
KW  - CAPILLARY ELECTROPHORESIS
KW  - SANGER SEQUENCING
KW  - MASSIVELY PARALLEL SEQUENCING BY
KW  - OLIGONUCLEOTIDE LIGATION AND DETECTION
N2  - Bisulfite sequencing is widely used for analysis of DNA methylation status (i.e., 5-methylcytosine [5mC]vs. cytosine [C]) in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by polymerase chain reaction (PCR)amplification of specific regions of interest that, overall, converts C?T (thymine)and 5mC?C and then capillary sequencing to measure C versus T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD)has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole enomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite conversion sample preparation to include a human genome-wide fragment library for SOliD. The current article features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment library conversion, which we refer to as Bis-PAGE, capillary-based size analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR)of randomly selected library fragments. smPCR/Capillary Sanger sequencing of approximately200 amplicons unambiguously demonstrated greater than 99
UR  - https://drive.google.com/file/d/1aIUBcX6mh7khfHyhIp55A4X_Gz0pwZDG/view?usp=drivesdk
ER  -