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Restriction enzymes information for exam conditions essay
ESSAY PLAN:
1. definitions of stuff
2. how it works
3. how it is used.
4. Conclusion: largely obsolete due to the rise of inexpensive DNA
sequencing technologies
Definitions
Restriction enzyme mapping - determining the order of fragments produced by
cutting a DNA molecule with a restriction enzyme.
RFLP - restriction fragment length polymorphism, a difference in the size of a
genomic DNA fragment produced by digestion with a particular enzyme. A useful
DNA marker. RFLPs Restriction Fragment Length Polymorphism are markers for
defined regions of the genome Used to track regions of the genome or as
markers to follow traits. Can be used to track diseases in a pedigree and discover
regions of the gnome where mutations might be.
Both to identify whether a particular mutation is present, and to determine
where in the genome mutations causing disease are located.
Restriction enzyme mapping – is the technique used for detecting RFLPs, need to
integrate this into the explanation below.
If we take a clone of DNA we can generate a map of the DNA by digesting using
restriction enzymes, and fractionating the resulting fragments on agarose gel
using electrophoresis, and determining their sizes by comparison with a set of
markers of known molecular weight that are run alongside the digest on the gel.
This technique can be make more informative using southern blotting, where the
fractionated DNA is transferred to a nylon membrane, and then specific
sequences can be detected and their length determined by interrogating the
membrane with a complementary DNA probe, which hybridises with the
fragments, and is labelled with a radioisotope.
Using RFLPs to diagnose disease:
Use of RFLPs is a powerful means of diagnosing genetic disease.
RFLPs: The sequence of the β Globin gene reveals that the amino acid altering
mutation also changes a restriction enzyme recognition site (DdeI- the
restriction enzyme- recognises the sequence CTNAG).
If the DNA from both a person with and without sickle cell anaemia is digested
with DdeI and then a southern blot is prepared, probed with a fragment of the
beta globin gene. DNA from normal indiviudals shows two fragments, as the DdeI
has been able to cleave at the restriction site, while DNA from sickle cell
individuals only shows one fragment of the gene- as the mutation has resulted in
the DdeI recognition site altering so that it is no longer recognised and cleaved
by the enzyme. This difference in restriction digest patterns is known as
Restriction Fragment Length Polymorphism (RFLP).
When a mutation, such as one that results in a genetic disease, causes a change in
DNA sequence that affects a restriction enzyme site, we can track this by
Southern blotting.
DISCOVER REGIONS OF THE GENOME WHERE MUTATIONS MAY BE LOCATED
RFLPs can also be used in tracking down the genes responsible for genetic
diseases. The location in the genome where the mutation is located can be
identified using linkage. 2 loci on a chromosome may be separated by
recombination, the further apart they are the more likely this is, but if the
markers are close together the chance they will be separated by recombination is
low.
If the mutant gene that causes the disease is located close to upstream sites for a
restriction enzyme, one of which is constant in the population, and another of
which is polymorphic, varying in its position perhaps due to variable length
repeat between the sites, then we can track the RFLP in a pedigree of disease
transmission. If the RFLP always segregates with the disease, showing a
similar pattern of inheritance, we infer it must be closely linked. This
indicates that the polymorphic restriction site is close to the affected gene,
and we can look in this region of the genome for candidate mutant genes
that cause the disease. This technique is more effective and successful
when attempting to find a gene in the entire genome, when a set of RFLPs
are scattered throughout the genome, and the polymorphism is present at
a reasonable frequency in the population, with large proportions of the
population having different fragment lengths.
USEFULNESSS:
This diagnosis method can even be used to identify heterozygous carriers of
recessive diseases, as in the case of sickle cell anaemia the signature Diagnostic
376bp band produced when the mutation is present will still be observed.
pre-natal diagnostic parental genetic testing.
Many genetic diseases may not generate useful RFLPs.
1. largely obsolete due to the rise of inexpensive DNA sequencing
technologies