Biology Reference
In-Depth Information
dark bands. The dark bands are primarily AT-rich regions and thus correspond to
most Q-bands.
C banding
requires a stringent extraction step that can result in
loss of chromosomal DNA. During C-banding, chromosomes are treated with a
strong base at a high temperature, incubated in a sodium citrate solution again
at high temperature, and stained in a concentrated Giemsa solution. C band-
ing extracts almost all of the non-C band chromatin, leaving only constitutive
heterochromatin, which usually contains rapidly reassociating repeated DNA
sequences (
Sessions 1996
).
Cytogenetic data provide information independent from morphological, bio-
chemical, or behavioral data for phylogenetic analyses. Cytogenetic data can
reveal differences or similarities that may not be obvious at the morphological
level. Chromosome size, shape, number, and ploidy levels can provide insights
into the genetic architecture of taxa. Banding studies reveal aspects of the struc-
tural organization of chromatin on individual chromosomes, whereas probes of
DNA sequences with
in situ
hybridization can reveal finer details of chromosome
anatomy in terms of spatial arrangement, and presence or absence of particular
kinds of DNA sequences.
12.4.3 Restriction Fragment Length Polymorphism (RFLP) Analysis
Restriction-enzyme analyses are versatile, providing information on the nature,
as well as the extent, of differences between sequences in nuclear or mitochon-
drial DNA (mtDNA) (
Dowling et al. 1996
,
Table 12.3
). RFLP analyses reveal varia-
tions within a species in the length of DNA fragments generated by a specific
restriction endonuclease. RFLP variations are caused by mutations that create or
eliminate recognition sites for the restriction enzymes.
RFLP analyses can be used effectively, and relatively economically, to analyze
clonal populations, heterozygosity, relatedness, geographic variation, hybrid-
ization, species boundaries, and phylogenies ranging in age from 0 to 50 mya
(
Table 12.3
). It is possible to analyze more loci per individual by RFLP analysis
than by DNA sequencing because RFLPs are less time-consuming and expensive,
although the information provided for each locus is less complete (
Dowling et al.
1996, Hall 1998
). Higher level systematics studies only rarely have used RFLPs.
More than 1400 restriction enzymes are known that cut DNA at a specific
position within a specific recognition sequence. See Chapter 5 for a discussion of
restriction digests, as well as
Brown (1991)
, and catalogs from a variety of com-
mercial producers. Most recognition sequences are 4-6bp long, although they
can be as large as 12 bp. The specificity of restriction enzymes means that a com-
plete digestion will yield a reproducible array of DNA fragments. Changes in the
