BIOL114 - Lecture 11 - Testing for Diseases

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  • Created by: Katherine
  • Created on: 17-03-16 10:39
What are the advantages of DNA testing?
Can test for specific alleles (predict severity), can test any cell at any stage of development ( prenatal or embryonic screening), can test for carrier status, high sensitivity of PCR (small sample required)
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Why might you need to conduct a DNA test?
To do neonatal screening - early detection and treatment can lead to improved prognosis e.g CF. For carrier screening for autosomal recessive disorders e.g CF, Predictive screening for late onset autosomal dominant disorders e.g. Huntingtons.
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What are other reasons for DNA testing?
Embryo pre-implantation screening (IVF) if high risk of affected child.
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What are the ways of obtaining foetal DNA?
Invasive foetal screening (Chorionic villus sampling amniocentesis) if high risk of affected child. Non invasive prenatal diagnosis (NIPD) uses blood sample from mother if high risk of affected child.
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What are the methods of genetic testing?
PCR amplification of mutant/ normal alleles. Do this to work out size differences and allel specific amplification. Fluorescent in situ hybridisation (FISH), Single nucleotide Polymorphism profiling, whole genome sequencing.
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What is the FISH technique used for?
Much larger chromosome deletions
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What is one disease that is diagnosed by PCR?
Huntington disease
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What is huntington disease caused by?
Increased number of CAG repeats in the HTT gene.
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How many repeats causes the gene to become toxic?
>35 - a normal allele has between 5 - 35 repeats
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Is huntington disease dominant or recessive?
Dominant - so just one mutant allele will cause the disease
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PCR can also be used to look for smaller changes, such as the mutation in...
CFTR. The deletion of 3 alleles at F508, in the CFTR. Because it is just a 3 base pair difference between the mutant and normal, the mutant is only 3 pairs shorter. So a polyacrylmide gel is used for this small scale fragementing.
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PCR can also be used to test for a particular base pair at a particular position, this is called:
Allele specific PCR.
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F
A primer for PCR will only work if there is a base pari formed at the 3' of the primer. DNA polymerase adds base pairs onto the 3' end. It will only do this if there is a perfect base pair between base at 3' end and template..
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How do you test for multi-gene disorders?
Identify high susceptibility alleles, which alleles result in a high chance of someone getting a disease. Certain combinations will give a higher susceptibility.
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What is a multi-gene condition?
Dseases caused by variation in several different genes
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What is the technique?
Looking at SNPS - Single nucleotide polymorphisms.
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Do SNP cause a disease?
Not necessarily. It just has to be close to the disease causing gene, or within the gene.
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With a SNP, what do you do?
YOu look in the population for a particular combination of a particular allele with a particular trait.
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What is this type of study called?
GWAS - Genome Wide Association Stidy
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How is this type of study conducted?
Genomes of many people with a certain genetic condition are tested for millions of SNPs (on a microarray), compared with unaffected individuals, statistically significant highe frequency amongst affected individuals = association.
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What are the tests designed to look for?
High risk alleles and the SNPs associated with them.
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How is SNP testing conducted?
We have primers that are designed to stop just before a particular known snp in a genome. This primer is hybridized to DNA from the patient. DNA polymerase is added with fluorescently labelled nucleotides, a nucleotide will be added.
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What is special about the nucleotide?
The nucleotides are similar to the ddNTPs and are dyed. The nucleotide complementary to the SNP will join to it and be a certain colour so identifiable.
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What does SNP detection occur on?
Arrays (glass microscope slides which have millions of different primers attached to them. Each point on the slie has a different primer). The chip is then scanned with a lazer and then the colour detected.
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How can SNP detection be commercialised?
23andMe - some private companies offer testing to identify likliness of developing a disease. and different traits
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What are the problems associated with SNP testing?
Weak association with risk (only slight increase) - usually environmental factors are more important. Unless BRCA1/BRCA2. They therefore have a very poor predictive value. The risk depends on combination of alleles from several genes.
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What are the problems with the karoytyping methods?
It works for large deletions/ rearrangements. But not anything smaller than 5million bp.
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What can we use to identify these smaller rearrangements?
FISH
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How does FISH work?
It uses a probe that is designed to match the region of the chromosome that might have been deleted or duplicated.
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What is the process of FISH?
You take the metaphase chromosomes (in the same way as a Karyotype) and spread them on the slide. Denature the DNA and hybridize the probe. If there is no deletion the probe will hybridize and you will see green.
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What happens if there is a deletion?
The probe won't hybridize and green fluorescence will be absent.
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Why is there a control in the FISH test?
Assists in the identification of the correct chromosome and it is there to ensure that the hybridization process is working properly.
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What is the advantage of using interphase cells rather than metaphase?
The whole analysis can be done very quickly - if you're using metaphase cells, you have to culture the cells, and that can take a few days to a week or 2. If you've done CVS, time is of the essence. Parents may wish for termination.
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What are the problems with current genetic tests?
They are restricted to one, or a few, disease alleles. They need some prior knowlege of alleles/region to be tested.
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In order to overcome these issues, what can be done?
Whole genome sequencing which could be used comparatively.
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What are the issues with whole genome sequencing?
High costs
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What are the aims of the NHS for genome sequencing this year?
To sequence rare congenital conditions and identify the mutations, to sequence cancer genomes to compare against normal ones, to look at pathogen genomes and patient genomes to determine infectious diseases.
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What is NIPD?
Non-invasive prenatal diagnosis
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What does NIPD rely on?
In a pregnant woman, nearly 6% of the DNA in her maternal serum comes from the foetus.
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What does this use to count numbers of copies of whole chromosomes (or individual alleles)?
Ultra-high throughput sequencing
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Theoretically, this could diagnose what?
All diseases from one blood sample.
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What has NIPD been able to diagnose?
Downs syndrome
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What does NIPD mean?
That people don't need to go through CVS or amniocentesis
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What are the ethical consequences of NIPD?
Increased number of abortions, prevention of disabled births, could lead to Eugenics (where do we draw the line?) It could lead to sex selection, a search for desirable traits or paternity testing in vitro which could lead to abortions.
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In NIPD how accurate is it?
If the value is close to 1, you can say with complete certainty thatthe baby will not have down syndrome, if it lies abotu the threshold value, you can be 97% sure that the foetus has down syndrome = this means that people are less likely to need cvs
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Why might you need to conduct a DNA test?

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To do neonatal screening - early detection and treatment can lead to improved prognosis e.g CF. For carrier screening for autosomal recessive disorders e.g CF, Predictive screening for late onset autosomal dominant disorders e.g. Huntingtons.

Card 3

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What are other reasons for DNA testing?

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Card 4

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What are the ways of obtaining foetal DNA?

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Card 5

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What are the methods of genetic testing?

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