John M. S. Bartlett.pdf - Bio-Nica.info

68 Bartlett
Table 2
Separation of Double-Stranded DNA:
What Percentage of Acrylamide?
Acrylamide (% w/v)
DNA size range
13.5 1000 –2000 bp
15.0 1180 –500 bp
18.0 1160 – 400 bp
12.0 1140 –200 bp
15.0 1125 –150 bp
20.0 1116 –100 bp
2.2.1. Nondenaturing PAGE Conditions
Although both TAE and TBE can be used for PAGE, we recommend use of TBE
because this generally provides sharper bands on low-percentage gels. Running gels in
1× TBE under low voltages (1–8 V/cm) will prevent denaturation of DNA caused by
heating of the gel. Although the electrophoretic mobility of double-stranded DNA
is inversely proportional to the log(fragment length), this relationship can be altered
by both GC content and sequence. For this reason, PAGE is not a reliable means of
sizing DNA fragments.
2.2.2. Denaturing PAGE
Denaturing page gels contain a denaturing agent (usually 6–8 M urea) that inhibits
base pairing in nucleic acids. DNA fragments are loaded after a brief heat denaturation
and electrophoresis at high voltages (ca 20 V/cm) ensures that high temperatures are
maintained throughout the electrophoretic process. Denaturing gels are most commonly
used to analyze sequencing reactions and to recover labeled DNA probes. As one
would predict from their common use in sequence determination, the motility of DNA
fragment under denaturing conditions is almost totally unaffected by sequence and
base composition.
2.2.3. Recovery of DNA from Polyacrylamide Gels
In our experience, recovery of DNA from polyacrylamide gels is vastly simpler than
recovery from agarose, largely because of the fact that much larger quantities of DNA
are loaded onto the gel. Elution overnight by diffusion can recover 70 to 80% of the
nucleic acid in Maxim–Gilbert’s solution.
2.2.4. Radioisotopic Detection and Quantitation
One of the major advantages of polyacrylamide gels is the ability to dry them
onto supports and produce autoradiographic images. These can either be analyzed by
densitometry or used as a template to allow direct counting of the bands of interest.
Although the latter approach is time consuming, it provides a highly quantitative
analysis of DNA species in (for example) quantitative PCR (3).