Biology Reference
In-Depth Information
number and size of fragments can occur by changes in DNA sequence by rear-
rangements (inversions, tandem duplication, and inverted duplication), or addi-
tion, deletion, or substitution of specific bases.
Once the DNA is digested with a restriction enzyme, the fragments produced
are sorted by size using agarose or polyacrylamide gel electrophoresis. DNA
fragments of known length are run on each gel to serve as an internal standard
and to allow the size of the experimental fragments to be estimated. The DNA
fragments in the gel are visualized by several methods, including staining with
ethidium bromide (if the DNA was previously amplified by the PCR), or by prob-
ing Southern blots with labeled probes. The detection technique used depends
on the amount of DNA present in the gel.
Staining with ethidium bromide is simple and cheap, but least sensitive. The
minimal amount of DNA in a band that can be detected by ethidium bromide is
≈
2 ng, so small fragments can be detected only if a large amount of DNA is pres-
ent. DNA probes can be end labeled by adding
32
P-labeled nucleotides to the
ends of DNA fragments produced by the restriction enzymes. Intensity of label-
ing is independent of fragment size and is more sensitive than ethidium bro-
mide (EtBr), with 1-5 ng of DNA easily visualized. If primers are available, DNA
can be first amplified by the PCR, cut with a restriction enzyme, and labeled by
ethidium bromide (PCR-RFLP).
If less DNA is available, radiolabeled DNA probes can be used to visual-
ize fragments. Southern-blot hybridizations are highly sensitive and picogram
quantities of DNA can be detected, although small fragments
<
50 bp in size are
more difficult to detect. Southern blots require a suitable probe with sufficient
sequence similarity to the target DNA that a stable hybrid can be formed at
moderate to high stringency. The use of probes from other species (heterolo-
gous probes) makes interpretation of results more difficult.
12.4.4 DNA and Genome Sequencing
Sequences of proteins, RNA, and DNA have been obtained during the past 40
years. The first sequence information was obtained from proteins in the mid-
1950s. RNA was sequenced in the mid-1960s, and DNA sequences were obtained
in 1975 after Sanger sequencing methods were developed. More recently,
NextGen sequencing methods have greatly reduced the time and costs of
sequencing genomes and the third-generation sequencing technologies promise
to further reduce costs and time, while increasing read lengths (see Chapter 7).
The use of the PCR makes DNA sequencing of single genes or several gene
fragments rapid and relatively inexpensive for systematic studies. Core facilities
