Characterization of high molecular mass linear polyacrylamide powder prepared by emulsion polymerization as a replaceable polymer matrix for DNA sequencing by capillary electrophoresis.
In a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long read-lengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al., Anal. Chem. 1996, 68, 3305-3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa, with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measurements to characterize the polymer, good batch-to-batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solutions containing 2% w/w of LPA made by emulsion polymerization were simple to prepare, resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied, allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read-length of more than 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.
Goetzinger
W
W
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
Kotler
L
L
Carrilho
E
E
Ruiz-Martinez
M C
MC
Salas-Solano
O
O
Karger
B L
BL
D000180:Acrylic Resins; D019075:Electrophoresis, Capillary / Q000379:methods*; D004655:Emulsions; D008970:Molecular Weight; D011108:Polymers; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
D000180:Acrylic Resins; D004655:Emulsions; D011108:Polymers; C016680:polyacrylamide gels