[Optimization of non-gel sieving capillary electrophoretic separation of DNA fragments of hundreds of base pairs].
Non-gel sieving capillary electrophoresis has been employed in the biological sciences for the size-based separation of macromolecules such as nucleic acids. In this paper, four factors i.e. electric field strength, capillary length, capillary diameter and hydroxy-propylmethylcellulose (HPMC) concentration were integratively evaluated to select the optimal condition of separating DNA fragments of a few hundreds of base pairs through orthogonal analysis. Conclusion was made through comprehensive analysis: better separation could be achieved in longer capillary, smaller inside diameter of capillary and less field strength. In practical application, effective separation in short time is important. We preferred to employ 8 g/L HPMC in coated capillary (37 cm x 75 microns i.d.) and electric field strength of 324 V/cm in separating DNA fragments of hundreds of base pairs. From sampling to getting results only a few more than ten minutes were needed in capillary electrophoresis. It needs less amount of sample (a few nanoliters) and shows higher sensitivity (0.1 pmol in UV detection) than polyacrylamide gel electrophoresis (PAGE). pBR322/Msp I fragments were completely resolved in both capillary electrophoresis and PAGE, and four-bases resolution was obtained. When the concentration of the PCR product is too low (< 20 mg/L), PCR reaction system is used as a negative control to make sure of the peak generated by the unpurified PCR products, avoiding interference made by PCR buffer and polymerase.
Ding
X
X
Laboratory of Genetic Analysis, National Center of Biomedical Analysis, Beijing, 100850.
Liao
J
J
Liu
X
X
Wang
Q
Q
Ma
L
L
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D053938:DNA Fragmentation; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D009696:Nucleic Acids / Q000032:analysis*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D009696:Nucleic Acids