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

Detection of Nucleic Acids 67
2.1.3. Loading Dyes
Loading dyes serve two purposes: They are usually dense and promote the settling
of the DNA to the base of the well. They usually contain dyes that migrate with the
DNA and can be visualized to monitor the process of electrophoresis. Care should
be taken, however, because the common dyes used (bromophenol blue and xylene
cyanol) have different motilities in different agarose products, with differing buffers
and differing gel percentages (see ref. 3).
2.1.4. DNA Dyes: Before or After?
Most dyes (ethidium bromide, SYBR green, etc.) that are used to stain DNA do
so by intercalating into the DNA sequence. As such they, of necessity, alter both the
structure and motility of the DNA. Although this can give spurious results, for many
applications (such as confirmation of PCR products before cloning or sequencing) this
is not a major issue. In these cases, the dye is often added to the loading buffer or gel
before electrophoresis. Where an accurate determination of product size is required,
such as in randomly amplified polymorphic DNA (RAPD) or allelotyping, products
should be separated in the absence of dye and the gel stained thereafter.
2.1.5. Recovery of DNA from Agarose Gels
Having identified the product on agarose gels, often there is a requirement to
sequence or otherwise analyze the product. Recovering DNA from agarose gels is a
simple procedure that can be attempted in many ways (1). It is important, however, that
the decision regarding recovery of DNA is taken before electrophoresis is attempted
as the choice of agarose is probably the most crucial factor in determining DNA yield
after extraction from agarose. Protocols for recovery of DNA from agarose can be
accessed from the web (see ref. 1 for a good range of methods).
2.2. Polyacrylamide Gel Electrophoresis (PAGE) of DNA
Casting and running polyacrylamide gels is undoubtedly more complex and problematic
than using agarose electrophoresis. Almost universally, polyacrylamide gels
are supported between glass plates and must be polymerized by a chemically catalyzed
reaction with the inherent problems caused by failed reagents from time to time.
However, PAGE has distinct advantages in terms of resolution, capacity, and purity of
DNA bands. The resolving power of PAGE is such that molecules of DNA with lengths
differing by as little as 0.2% (2 bases/Kb) may be separated (although some novel
agarose preparations can approach this for smaller fragments). PAGE has a higher
capacity for DNA than agarose, with up to 10 µg of DNA per lane being applied
without compromising results. DNA recovered from PAGE is extremely pure and has
been used for microinjection into mouse embryos (2).
PAGE is equally applicable to separation of double-stranded or single-stranded
DNA (under denaturing conditions); however, the size range of fragments that can
be separated is more restrictive than for agarose because of the fragile nature of low
percentage acrylamide gels (see Table 2).
PAGE gels are crosslinked using a varying ratio of bis-acrylamide in the monomeric
solution; generally, the ratio of acrylamide to bis-acrylamide is around 301.