The effects of field inversion electrophoresis on small DNA fragment mobility and its relevance to DNA polymorphism research.
The electrophoretic mobility of several DNA size markers of molecular lengths from 1.23 x 10(2) to 2.36 x 10(4) base pairs has been investigated in gels of 1% and 1.3% agarose (w/v) by field inversion gel electrophoresis (FIGE), in vertical slabs. Pulsing times studied were in the range of 0.3/0.1 ms to 300/100 ms. (Pulsing times are given in milliseconds, as X/Y, where X is the forward pulsing time and Y is the reverse time.) FIGE differentially retards DNA migration: this effect is more marked for shorter pulsing times, and varies as a function of molecular length of the DNA fragment, down to fragments as small as about 1.7 kb, with the pulsing times we used. Several FIGE conditions were found which generate improved resolution of DNA fragments in different size ranges. DNA separation improves by more than a factor of two for fragments of 23.1-9.4 kb (with 300/100 ms pulsing) and for fragments of 4.4-2.0 kb (3/1 ms). FIGE does not seem to have a marked resolution enhancing effect on DNA sized from 9.4-4.4 kb. An example of improved detection of closely spaced bands on Southern blots is shown.
Gejman
P V
PV
National Institute of Mental Health, Clinical Neurogenetics Branch, Bethesda, Maryland 20892.
Sitaram
N
N
Hsieh
W T
WT
Gelernter
J
J
Gershon
E S
ES
D015139:Blotting, Southern; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted; D012150:Polymorphism, Restriction Fragment Length