8B - Gene probes and medical diagnosis

Question 1

What method do we use to locate the exact DNA sequence of a mutation/disorder?

Answer 1

Using gene probes and DNA hybridisation.

Question 2

What are DNA probes?

Answer 2

Short single-strands of DNA that has a label attached to make it identifiable.

Question 3

What do DNA probes have a specific base sequence that is complementary to?

Answer 3

The base sequence of part of the target allele.

Question 4

What id dsDNA?

Answer 4

Double-stranded DNA.

Question 5

What will happen due to the DNA probes having a specific base sequence that is complementary to the base sequence of part of the target molecule?

Answer 5

DNA probe will bind (hybridise) to the target allele if it’s present in a sample of DNA.

Question 6

How do DNA probes work (briefly)?

Answer 6

dsDNA being tested is separated into 2 single strands which are then mixed with the probe which binds to the complementary sequence on one of the strands (DNA hybridisation).

Site can be identified depending on the type of probe used.

Question 7

What are the 2 types of labelled probes?

Answer 7

Radioactively labelled probes and fluorescently labelled probes.

Question 8

What are radioactively labelled probes?

Answer 8

Made up of nucleotides with isotope 32P.

Identified using X-ray film exposed by radioactivity.

Question 9

What are radioactively labelled probes identified by?

Answer 9

X-ray film

Question 10

What are fluorescently labelled probes identified by?

Answer 10

UV light

Question 11

What are fluorescently labelled probes?

Answer 11

Emit light (fluoresce) under certain conditions.

Question 12

What is DNA hybridisation?

Answer 12

When a section of DNA (or RNA) is combined with a single stranded section of DNA.

Question 13

What has to happen before a section of DNA (or RNA) is combined with a single stranded section of DNA - in DNA hybridisation?

Answer 13

The 2 strands must be separated:

• Heat DNA until separated (denaturation).
• When cooled, complementary bases recombine (anneal).
• If other complementary sections of DNA are present, they are just as likely to anneal with one separated DNA strand as the 2 strands are with one another.

Question 14

How are fluorescently labelled probes used?

Answer 14

• Sample of DNA digested into fragments using restriction enzymes and separated using electrophoresis.
• Separated DNA fragments then transferred ti a nylon membrane and incubated with the fluorescently labelled DNA probe.
• If allele is present, the DNA probe will hybridise it.
• The membrane is then exposed to UV light and if the gene is present there will be a fluorescent band.

Question 15

What can fluorescently labelled probes be used as part of?

Answer 15

A DNA microarray

Question 16

What can a DNA microarray do?

Answer 16

Screen lots of genes at the same time.

Question 17

Explain how fluorescently labelled genes can be used as part of a DNA microarray

Answer 17

• Sample of fluorescently labelled human DNA washed over the array.
• If the labelled human DNA contains any DNA sequences that match any of the probes, it will stick to the array.
• The array is washed, to remove any labelled DNA that hasn’t stuck to it.
• The array is then visualised under UV light (any labelled DNA attached to a probe will show up - fluoresce).
• Any spot that fluoresces means that the person’s DNA contains that specific allele. E.g. if the probe is for a mutated allele that causes a genetic disorder, this person has the allele.

Question 18

What is a DNA microarray?

Answer 18

A glass slide with microscopic spots of different DNA probes attached to it in rows.

Question 19

What do you need to produce a DNA probe?

Answer 19

First need to sequence the allele that you want to screen for.

You then use PCR to produce multiple copies of part of the allele - these are the probes.

Question 20

What can genetic screening be used to do?

Answer 20

• Help identify inherited conditions.
• Help determine how a patient will respond to specific drugs.
• Help identify health risks.

Question 21

How can genetic screening help identify inherited conditions?

Answer 21

People with family history can be screened for mutated alleles that cause the disease.

All newborn babies offered screening for cystic fibrosis.

Question 22

What do people who are found to be at risk of a disease through genetic screening go through?

Answer 22

Genetic counselling.

Question 23

How can genetic screening helping to identify health risks be useful?

Answer 23

if a person knows they have the alleles it might help them to make choices that could reduce the risk of the disease developing.

Question 24

What are the concerns with genetic screening?

Answer 24

Some are concerned it may lead to discrimination by insurance companies and employers if people are known to have a high risk of developing a condition.

Question 25

What can the results of screening be used for?

Answer 25

Genetic counselling

Question 26

What is genetic counselling?

Answer 26

Advising patients and their relative about the risks of genetic disorders.

Question 27

What does genetic counselling involve?

Answer 27

Advising people about screening (e.g. looking for mutated alleles if there's a history of cancer) and explaining the results of a screening. Screening can help to identify if someone is the carrier of a mutated allele, the type of mutated allele they're carrying (indicating the type of genetic disorder or cancer) and the most effective treatment.

Question 28

What is genetic counselling used for if the result of a genetic screening are positive (an individual has the mutation)?

Answer 28

Used to advise the patient on the options of prevention or treatment available.

Question 29

How can genetic counselling be used if a women has a history of breast cancer?

Answer 29

- May help her decide whether or not to be screened for known mutations that can lead to breast cancer. - If she is screened and the result is positive, genetic counselors might explain that a women with the mutated BRCA1 gene has a 50 to 85% chance of developing breast cancer in her life. - It could also help her decide if she want to take surgical steps to remove the risk of breast cancer developing (by having a mastectomy).

Question 30

How can genetic counselling be used for sickle cell?

Answer 30

Sickle-cell anaemia is a recessive genetic disorder caused by a mutation in the haemoglobin gene. A couple who are both carriers of it may like to have kids. They may undergo genetic counselling to help them understand their chances of having a child with sickle-cell (1 in 4). Genetic counselling also provides unbiased advice on the possibility of having IVF and screening their embryos for the allele, so embryos without the mutation are implanted in the womb. It could also provide information on the help and drugs available if they have a child with sickle-cell anaemia.

Question 31

How can genetic screening be used in personal medicine?

Answer 31

Different people respond to drugs in different ways and this is determined by your genes. Therefore personalised medicines that are tailored to an individual's DNA could be used. The idea is that if doctors have your genetic information, they can use it to predict how you will respond to different drugs and only prescribe the ones that will be most effective for you.

Question 32

Explain how personal medicines can be made

Answer 32

- Identification of genes and biomarkers. - Allocation of resources to prevent/detect phenotype in those with genotype at risk. - Selection of individualised therapies for affected individuals. - Measurement of circulating biomarkers to monitor the response to prevention or therapy.