High-resolution capillary electrophoretic analysis of DNA in free solution.
Capillary electrophoretic separations of double-stranded DNA fragments in the size range of 20-2200 base pairs were achieved in less than 20 min with the use of a Tris-borate buffer containing hydroxyethylcellulose. Analyses were carried out in both uncoated and phenylmethyl-coated capillaries of fused silica with internal diameters of 50 and 100 microns, respectively, and an effective column length of 50 cm. The addition of ethidium bromide resulted in an improved resolution of double-stranded DNA fragments, thereby permitting even the separation of fragments differing only 1-2 base pairs in length. Moreover, resolution was found to be linearly proportional to the size of the cation used to adjust ionic strength Cs+ greater than RB+ greater than K+ greater than Na+ greater than Li+. However, the analysis times also increased with increasing cation size due to a decrease in electroosmotic flow. Elution order was verified by spiking restriction digests with slab gel electrophoretically purified components. Subsequently, the described system was applied to the detection and quantitation of an mRNA transcript of the androgen receptor, which had been amplified by polymerase chain reaction and purified by size-exclusion chromatography to avoid peak broadening due to conductivity differences between sample and running buffer. Since the actual amount of DNA introduced into the capillary cannot be defined, molar ratio-peak area ratios of the polymerase chain reaction product to various restriction fragments of known concentration were used to determine the amount of amplified DNA. The coefficient of variation was as small as 3.4% and the results were in good agreement with a spectrophotometric assay.
Nathakarnkitkool
S
S
Institute of Radiochemistry, University of Innsbruck, Austria.
Oefner
P J
PJ
Bartsch
G
G
Chin
M A
MA
Bonn
G K
GK
D001483:Base Sequence; D002478:Cells, Cultured; D002482:Cellulose; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification; D012996:Solutions
D009841:Oligonucleotides; D012333:RNA, Messenger; D012996:Solutions; D002482:Cellulose; D004247:DNA; D004262:DNA Restriction Enzymes; D004996:Ethidium