Temperature and pH studies of short tandem repeat systems using capillary electrophoresis at elevated pH.
The DNA secondary structure can affect the migration time and precision of DNA separations in the physical gels used in capillary electrophoresis (CE). To counteract these effects, DNA typing is performed using elevated temperatures (60 degrees C) and high concentrations (7 M) of urea. These conditions affect the precision and lifetime of the analysis. To better understand the effects of these conditions on the reproducibility of DNA migration, we examined the effects of temperature and pH on short tandem repeat (STR) analysis using the PE/ABI 310 Genetic Analyzer. Separations were performed using the Profiler + multiplex system, a set of coamplified STRs with a 4-base repeat motif, labeled at the 5'-end using fluorescent dyes. The analytical separations were obtained using a commercial buffer at pH 8 and an experimental buffer consisting of 3% hydroxyethylcellulose at pH settings ranging from 8-12. Multichannel laser-induced fluorescence detection was used. Temperatures were examined from 30-70 degrees C. The results demonstrate the fact that highly efficient separations can be carried out at alkaline pH. In addition, improvements in temperature stability were seen when compared to results at lower pH. However, high concentrations of urea were found to be necessary to achieve optimal resolution.
Nock
T
T
Department of Chemistry and Biochemistry, Athens, OH 45701, USA.
Dove
J
J
McCord
B
B
Mao
D
D
D000483:Alleles; D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis / Q000737:chemistry; D016208:Databases, Factual; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009691:Nucleic Acid Denaturation; D015203:Reproducibility of Results; D020080:Tandem Repeat Sequences; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D014508:Urea; D004247:DNA