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3.2.8b
FUNCTION IN ORDER TO BETTER UNDERSTAND ORGANISM FUNCTION ...
MEDICAL DIAGNOSIS
Introduction
Many human diseases have a genetic origin often in the form of a mutation.
For example sickle cell anaemia which may have mixed benefits and advantages to
the carrier of the gene.
Recombinant DNA technology (3.2.8a) allows the diagnosis of genetic disorders.
DNA probes and DNA hybridisation are procedures that assist in the diagnosis and
treatment of genetic disorders.
to the exposed bases in a process known as DNA hybridisation ... the site of
attachment can then be identified using the label either by autoradiography or
fluorescence.
Gene probes can only be produced is the gene that we want to find has a known
sequence of bases...
DNA sequencing
One method of determining the sequence of bases on a gene is Sanger sequencing.
Stage 1:
Four tubes are set up each containing:
A* T* C* G*
Nucleotides A, T, C, G Nucleotides A, T, C, G Nucleotides A, T, C, G Nucleotides A, T, C, G
Labelled primers Labelled primers Labelled primers Labelled primers
DNA Polymerase DNA Polymerase DNA Polymerase DNA Polymerase
Single stranded target Single stranded target Single stranded target Single stranded target
DNA to be sequenced DNA to be sequenced DNA to be sequenced DNA to be sequenced
Stage 2
The fragments are separated by gel electrophoresis.
The DNA fragments are placed on an agar gel and a voltage applied across it.
Larger fragments move more slowly and so over time, the smaller fragments move a
further distance than the larger ones.
In this way the DNA fragments of different lengths are separated.
A sheet of photographic film that is sensitive to radioactivity is placed over the plate.
The exposure of the film shows the location of the DNA fragments on the plate.
The plate is read from the bottom up as the shortest fragment will have travelled
the furthest. Eg the DNA base sequence below is CAAGTT
This method only works well for sequences of less than 500 nucleotides.
For larger genomes, then restriction mapping is used.
Restriction Mapping
Restriction mapping is a process which cuts up DNA into fragments using a “cocktail”
of different restriction enzymes to produce arrange of fragments of varying sizes.
The fragments are produced by cutting with pairs of enzymes.
These fragments can then be separated by gel electrophoresis.
The distance between the recognition sites can then be determined by the pattern
of fragments produced.
Restriction mapping is best shown by example.
A plasmid of 100kb is cut using three different restriction enzymes.
Each cut produces two fragments whose size can be estimated using gel
electrophoresis.
This information can now be used to construct a restriction map of the plasmid.
Genetic Screening
Genetic disorders such as sickle cell anaemia are the result of genetic mutations.
Mutation forms mutant alleles.
If there is a family history of certain diseases it is important to screen potential
parents to determine the probability that an offspring will carry the mutated gene.
A genetic counsellor can then advise on the implications of having children based
on their family history.
Screening procedures
1. The sequence of nucleotides on the mutated gene is determined by DNA
sequencing and stored in DNA libraries.
2. A fragment of the DNA with a complementary sequence is produced.
3. A DNA probe is formed using radioactively labelled nucleotides.
4. PCR is used to make many copies of the probe.
5. The probe is added to single-stranded fragments of DNA from the test
subject.
6. If the mutant allele is present, the probe will bind using specific base pairing.
7. Exposure to a photographic film will determine whether the labelled probe is
present and so indicate where the mutant allele is present in the test subject.
Detection of oncogenes
Oncogenes are responsible for cancer.
Cancer can develop as a result of mutations that prevent the tumour suppressor
gene from inhibiting cell division.
Both alleles must have the mutated oncogene for cancer to develop.
Individuals who inherit one mutated oncogene are at greater risk of developing
cancer.
Screening can identify such individuals at risk and counselling can then give advice
on lifestyle and possible treatment protocols.
Genetic counselling
This is a form of social work which gives personal advice regarding the family
histories of genetic disorders.
For example, two potential parents who have a family history of sickle cell anaemia
may both be carries which gives a 25% chance that an offspring would be
homozygous for the condition, and the implications that this carries.
Genetic counselling is linked with screening.
GENETIC FINGERPRINTING
The genome of an organism contains many repetitive non-coding regions.
The statistical chances of two people carrying the same regions are very low.
The process follows five main processes.
1. Extraction; DNA is extracted from cells and amplified using PCR.
Specimen
June 2007
January 2009
January 2008
Specimen
January 2009