Biology Reference
In-Depth Information
discusses the DNA marker techniques and strategies that the sunflower
community adopted for genome mapping.
3.3.1 RFLP
RFLP (restriction fragment length polymorphism) is the first DNA marker
technique developed for genome mapping in humans (Botstein et al. 1980).
This technique detects the gain or loss of a restriction enzyme recognition
site and the insertion or deletion of a DNA fragment between two restriction
sites. The RFLP assay includes isolating high molecular weight DNA samples
from experimental individuals, digesting the DNA with a restriction
endonuclease, electrophoresis of an agarose gel to separate the DNA
fragments according to their size, blotting the DNA fragments onto a nylon
membrane, hybridizing the membrane with a labeled DNA probe that will
only bind to a specific sequence that is complementary to the probe, and the
detection of the hybridization signals. The probe is a cloned fragment from
a genomic or cDNA library. In the early days of RFLP analysis, the
hybridization signals were detected with
32
P labeled probes. It would often
take 2 or 3 weeks to complete a single round of hybridization. In the mid-
1990s, the advent of enzyme-linked probes and chemiluminescent detection
expedited the process dramatically and enabled one round of hybridization
to be completed within about a week. Even so, RFLP remained a slow, labor-
intensive technique and required a relatively large amount of high quality
DNA for the experiment. However, RFLP produced repeatable, excellent
results and the markers are highly informative. It was RFLP that
revolutionized the genome mapping effort for many economically important
animal and plant species.
The implication of RFLP to sunflower genome mapping was first
reported by the USDA-ARS Sunflower Research Unit (Jan et al. 1992). The
involvement of a private party in the map construction not only delayed
slightly the publication of the map, but also impeded distribution of the
probes to other research laboratories. The first sunflower RFLP marker map
was published in the mid-1990s (Barry et al. 1995; Gentzbittel et al. 1995).
3.3.2 RAPD
It is necessary to talk briefly about polymerase chain reaction (PCR) here
since the next several marker techniques are PCR-based. PCR is one of the
most powerful and revolutionary procedures used today to analyze DNA
sequences. It is not an exaggeration to say that the impact of this simple
procedure on modern biology is beyond description. PCR is based merely
on the unique biochemistry of DNA replication and uses a thermostable
DNA polymerase to amplify a DNA fragment that is flanked by known
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