aqa 358b gene technology biol5

GENE CLONING TECHNOLOGIES ALLOW STUDY AND ALTERATION OF GENE FUNCTION IN ORDER TO BETTER UNDERSTAND ORGANISM FUNCTION ...

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1 Copyright © Dr Robert Mitchell 2010

Biology Tutorials 40 Higher Bridge Street, Bolton, Lancashire, BL1 2HA www.biologytutorials.co.uk 01204-432447

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.

DNA probes and DNA hybridisation

A DNA probe is a short single stranded DNA that is complementary to a target gene and has a label attached to it to make it identifiable.

Labels can be radioactive (e.g. using 32P that can be identified using a photographic plate) or fluorescent which emit light when excited using specific wavelengths.

DNA probes can identify particular genes by being complementary to the portion of DNA sequence that makes up part of the gene whose position we want to find.

Flow chart ... The DNA under test is treated to separate the strands which are then mixed with the probe ... if the target sequence is present, the DNA probe will attach


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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 DNA to be sequenced




Note that the nucleotide* is a terminator nucleotide that is modified to prevent it from attaching to the next base in the sequence when they are being joined.

When binding takes place either the normal or the modified nucleotide can bind. And this can take place randomly either early on in the sequence or middle or end.

This results in the production of different length DNA fragments all of which have the terminal nucleotide as the modified one either A*, T*, C* or G*.

These fragments can be identified as they have the labelled primer attached to the other end.

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.


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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.


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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.


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2. Digestion; Restriction endonuclease enzymes are selected to cut up the DNA into fragments.

3. Separation: The fragments are separated according to size by gel electrophoresis. The gel is made alkaline to separate the double strands. The single-strands are then transferred to a nylon membrane by Southern blotting (nylon membrane is laid over the gel ... covered with sheets of absorbent paper to draw up the liquid with the DNA which transfers it to the membrane ... Fragments are then fixed with UV light).

4. Hybridisation: labelled DNA probes then bid o the core sequences. Many different probes are used to ensure all the fragments are recognised.

5. Development: A photographic sheet is then placed over the sheet and the x-rays emitted by the radioactive probes develop the film and generate bands corresponding to the positions of the DNA fragments.

Uses of genetic fingerprinting

Forensic science to determine the extent to which an individual is connected to a crime.

Paternity testing (Jeremy Kyle show!)

Genetic variability assessments for conservation purposes.

Selection of breeding pairs for endangered species to ensure the mates are not familiarly related.


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Specimen


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June 2007


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January 2009


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January 2008


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Specimen


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January 2009

aqa 358b gene technology biol5

Documents

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