DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures.
Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80 degrees C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N-dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (PDEA30). DNA-polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70 degrees -75 degrees C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50 degrees C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60 degrees C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50 degrees C. Molecular masses of these polymers were determined by tandem gel permeation chromatography-multiangle laser light scattering method (GPC-MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.
He
Hui
H
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
Buchholz
Brett A
BA
Kotler
Lev
L
Miller
Arthur W
AW
Barron
Annelise E
AE
Karger
Barry L
BL
D000178:Acrylamides; D000180:Acrylic Resins; D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D011108:Polymers; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions; D013696:Temperature; D013997:Time Factors; D000099166:Polyacrylamides
D000178:Acrylamides; D000180:Acrylic Resins; D004247:DNA; D011108:Polymers; D012996:Solutions; D000099166:Polyacrylamides; C429790:poly(N,N-dimethylacrylamide); C429791:poly(N,N-dimethylacrylamide-co-N,N-diethylacrylamide)