Biology Reference
In-Depth Information
specific activity of the label. Specific activity refers to the amount of radioactiv-
ity per milligram of probe DNA. The specific goals of the project will determine
which labeling technique is used. Detailed protocols are available in a variety of
laboratory manuals and kits. The safe use of radioactivity for labeling requires
that the user obtain specific training in handling procedures and disposal.
Nonradioactive probes using biotin and chemiluminescent labels are available
in kits and are safer for novices to use. These labels may be sufficiently sensitive
for most applications. However, if very small amounts of DNA or RNA are being
screened, radioactive probes may be most sensitive.
6.7.1 Synthesis of Uniformly Labeled DNA Probes by Random Primers
Short oligonucleotides can serve as primers for DNA synthesis by DNA polymer-
ases on single-stranded templates. If the primers used are random in sequence,
they will form hybrids at many different locations along the template strand
so that the strand being synthesized will incorporate a labeled dNTP randomly
along its length. If reverse transcriptase is used for synthesis, the template can
be RNA. If DNA is the template, then the Klenow fragment of DNA polymerase I
is used.
6.7.2 Synthesis of Probes by Primer Extension
Primer extension is used to synthesize probes from denatured ds DNA. It can be
used to produce probes from denatured, closed circular DNA or from denatured,
linear ds DNA. The purified DNA is mixed with random primers, denatured by
boiling, and labeled dNTPs and the Klenow fragment of DNA polymerase I are
added to carry out synthesis of the probe. Random primers anneal to the dena-
tured DNA and the product is synthesized by primer extension. Probes prepared
by random priming are usually 400-600 nt.
6.7.3 End-Labeled Probes
A variety of methods are available to introduce labels at either the 3
′
or 5
′
ends
of linear DNA. Usually, only a single label is introduced at one end of the mol-
ecule. Both DNA and RNA can be end-labeled. The advantages to end labeling
are that the location of the labeled group is known and very small fragments of
DNA can be labeled, including restriction-digest fragments.
6.7.4 Single-Stranded Probes
Single-stranded DNA, cDNA, or RNA probes have an advantage over ds probes
because more probe is available to hybridize with the nucleic acid of interest.
