Molecular Genetics III

Question 1

PCR - RFLP

Answer 1

• Mutations affecting a restriction site
• Once product is made after PCR, a restriction digest is performed
• Mutation must create or destroy a restriction site
• PCR is quick and inexpensive

Question 2

ASO - PCR (with blotting)

Answer 2

• Single base pair changes
• 2 probes, one looking for mutated allele and the other looking for the normal allele. Each hybridization reaction performed separately.
• Used for known common mutations (CF Δ508, hemochromatosis, sickle cell)
• Less useful if the disorder exhibits allelic or locus heterogeneity
  
  • test doesn’t guarantee that the individual isn’t a carrier for an untested mutation bc of allelic heterogeneity

Question 3

PCR sizing

Answer 3

• Size of PCR product
  
  • triplet repeat expansion disorders
    
    • Huntington’s
    • Myotonic dystrophy
    • Fragile X
• A primer pair that flanks (down stream and upstream) the expansion region

Question 4

reverse transcription (RT) PCR

Answer 4

• expression levels of mRNA
• First the mRNA is converted to cDNA then primer pairs are used to amplify the cDNA
• gene expression = mRNA

Question 5

FISH

Answer 5

• Submicroscopic deletions
  
  • can’t be seen by G-banding
• Chromosome specific unique sequence probes OR gene specific probe
• Used to detect DiGeorge syndrome
• Only gives data for what you are FISHing for, doesn’t query the entire genome

Question 6

karyotypes (G-banding)

Answer 6

• Chromosomal ploidy status & rearrangements insertions and deletions greater than 5Mb - 10 Mb
• stains AT rich regions dark bands and GC regions light
• Detects aneuplodies (trisomies) and other large changes

Question 7

SKY FISH

Answer 7

• chromosomal rearrangements (translocations)
  
  • Philadelphia Translocation
• each chromosome gets colored differently
• need to identify the chromosomal origin of rearranged chromosomes
• doesn’t detect small deletions
• SKY FISH gives info. that G-banding might miss
  
  • can help to identify the origin of a rearranged chromosome, but can also miss info seen by G-banding such as a deletion on chromosome

Question 8

array CGH

Answer 8

• detection of DNA sequence copy number changes throughout the genome in a single hybridization experiment
• probes are fixed on to a chip and shredded genomic DNA is allowed to hybridize
• CNV (copy number variance) includes trisomy conditions; also useful for cancer
• Array CGH is first tier test for children with developmental delay and mental impairment
• Array CGH may query the entire genome in a single test
• can’t detect balanced rearrangements
  
  • pitfalls: what’s normal? significant?

Question 9

SNP chip

Answer 9

• single base pair changes, polymorphisms and point mutations
• probes are fixed on to a chip and shredded genomic DNA is allowed to hybridize
• Searches 1000s of probes, can test for susceptibility to adverse drug reactions
  
  • CYPs
• Can detect uniparental disomy especially if the parents have had an SNP chip
  
  • PWS
  • Angelman Syndrome

Question 10

cDNA expression arrays

Answer 10

• gene expression as compared to a reference
• cancer profiling