Intercomparison of DNA sizing ladders in electrophoretic separation matrices and their potential for accurate typing of the D1S8O locus.
The 100-1,000 basepair size typical of PCR-amplified DNA fragments demands high resolution electrophoretic gels for the adequate characterization of small differences among samples. We have studied the behavior of a number of commercial sizing ladders in three classes of separation systems: polyacrylamides with discontinuous buffer, proprietary acrylamides with continuous buffer, and agarose-like materials with continuous buffer. None of the ladders examined perform adequately in any of these systems using vendor-supplied nominal ladder component basepair sizes. All ladders successfully typed D1S8O alleles after calibration with the allelic ladder (replacing the nominal size values with the least squares estimate of allele/matrix-specific apparent sizes). Some ladders and matrices are qualitatively better than others. No one ladder proved consistently better than others; a polyacrylamide gel with ribose modifier provided the most precise results in this study. Appropriately calibrated electrophoretic apparent sizes must be used for results to be validly exchanged among laboratories. Appropriate allelic ladders or a well defined subset of known alleles can serve as the calibration system.
Kline
M C
MC
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Redman
J W
JW
Reeder
D J
DJ
Duewer
D L
DL
D000178:Acrylamides / Q000737:chemistry*; D000483:Alleles; D004247:DNA / Q000145:classification*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D005819:Genetic Markers; D006801:Humans; D012091:Repetitive Sequences, Nucleic Acid; D012685:Sepharose / Q000031:analogs & derivatives*
D000178:Acrylamides; D005782:Gels; D005819:Genetic Markers; D004247:DNA; D012685:Sepharose