title: Capillary DNA sequencing: maximizing the sequence output.
abstract: Like most other DNA sequencing core facilities, one of our continuing goals is to improve our sequence output without substantially adding to cost. To minimize sample-to-sample variability in template DNA concentration, we implemented the rolling circle amplification (RCA) procedure for preparing our DNA templates. In addition to saving time and reducing the number of steps in template DNA preparation, the RCA method has the potential to normalize the DNA concentration in samples that can be sequenced directly without additional purification. In the present study, we used RCA-generated templates to test a recently reported procedure that increased sequence quality by resuspending the sequenced products in low concentrations of agarose before capillary electrophoresis (CE) on a MegaBACE 1000 platform. Although we did not obtain the expected result using the specified procedure, a modification resulted in up to 60% increase in total sequence yield per sample plate. A combination of agarose and formamide-EDTA in the resuspension solution enabled us to generate long-read and high-quality sequences for more than 38,000 templates with minimal additional cost.
authors.affiliation: The Biotechnology Program, University of Florida, Gainesville, FL 32610-0156, USA. ernie@biotech.ufl.edu
authors.forename: Ernesto C
authors.initials: EC
authors.lastname: Almira
authors.forename: Nedka
authors.initials: N
authors.lastname: Panayotova
authors.forename: William G
authors.initials: WG
authors.lastname: Farmerie
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004270:DNA, Circular / Q000235:genetics; D004492:Edetic Acid; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005559:Formamides; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions; D013698:Templates, Genetic
chemical_list: D004269:DNA, Bacterial; D004270:DNA, Circular; D005559:Formamides; D012996:Solutions; C031066:formamide; D004492:Edetic Acid