Genetic testing for disease

• 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)
• Methods of genetic testing

  • PCR amplification of mutant/normal alleles
  • This can be Size differences or Allele-specific amplification
  • Fluorescent in situ hybridisation (FISH)
  • Single Nucleotide Polymorphism (SNP) profiling
  • Whole genome sequencing
• Diagnosing huntingtons using PCR based on size repeats

  The mutant allele has >35 repeats and so produces a heavier band.

  1. Extract DNA
  2. PCR to amplify using primers that bind either side of the repeat
  3. Separate using polyacrylamide gel electrophoresis
• Cystic fibrosis detected using size difference in the CFTR repeat

  The mutant allele has one less codon. And so the gene size decreases from 98 to 95.

  Amplify using PCR and primers that flank either side of the sequence.

  Seperate using polyacrylamide gel electrophoresis

  
  

  Carriers will have both a 98 and 95 gene size.

  Sufferers will have only the 95 gene size.
• Allele-specific PCR

  Test for base at a particular location in the DNA
• Why is polyacrylamide used over agarose

  It has a higher resolution.

  If agarose was used the small sections of DNA would run off the gel.
• Describe allele-specific PCR to see if the mutant allele is present.

  The primer will stop where the mutation is and is complementary to the mutant allele.

  If the primer can be extended using PCR then they contain thee mutant allele.

  If it cannot be then they don't contain the mutant allele.
• Describe how SNPs are used in multi-gene disorder detection

  They use statistics to decide if the a version of a SNP is associated with a disease causing allele.
• Genome-Wide Association Studies (GWAS)for SNP association determination

  • Genomes of many people with the disease are tested for SNPs using a microarray
  • Compared with unaffected individuals
  • Statistically significant higher frequency amongst affected individuals = association
  • Design tests to look for high risk alleles which are associated with a high risk of disease
• SNP detection

  Microarrays contain wells in which probes are bound. They are complementary to the sequence just upstream of the SNP.

  
  

  Add fluorescently labelled nucleotides and amplify using PCR. Depending on the colour produced then you know what the SNP is.
• Problems with SNP testing

  • Weak association with risk (exceptions are BRCA1/BRCA2)
  • Poor predictive value
  • Environmental factors very important
  • Risk depends on combination of alleles from many genes
• Diagnosing chromosomal disorders

  This is done by culturing cells to enter metaphase and then hybridizing with complementary DNA in situ.

  
  

  Or you can use cells without culturing as they would be in interphase.
• Describe the different things you need to stain in FISH

  You use a red control stain to show that the primers are annealing.

  The DNA stains blue.

  The loci on the chromosome of interest stains green.
• Screening by whole genome sequencing

  • Use next generation, high-throughput sequencing technologies
  • Can detect all possible alleles in all
  • Can be used to detect chromosome abnormalities
• Genome sequencing in the NHS

  They were looking to identify mutations for rare diseases.

  Compairing the genome of cancer and non-cancer patients.
• Non-invasive prenatal diagnosis (NIPD) by sequencing

  • Relies on free foetal DNA (or RNA) in maternal serum
  • Uses high throughput sequencing to count number of copies of whole chromosomes (or individual alleles)
• Describe allele-specific PCR to see if the normal allele is present.

  The primer will stop where the mutation is and is complementary to the normal allele.

  If the primer can be extended using PCR then they contain the normal allele.

  If it cannot be then they have the mutant allele.