Genome Analysis Methods Flashcards
what are the first steps for genome analysis
do you know the gene in question
no - whole genome analysis = gene (chromosome dosage) - genome sequence
yes - do you know the mutation to test for
(no - gene sequence, yes - specific assay)
what is the general hybridisation principle for tests
two DNA molecules anneal into duplex only if their sequences are complimentary
mismatched bas pairs destabilise the duplex and allow discrimination between mutant vs normal strands
what is PCR
amplification of DNA in vitro (Kary Mullis)
synthesis of large amounts of DNA from small starting quantities
describe the process of PCR
primer (oligonucleotides) start the process and dna polymerase used monomers for new strands
step 1 - heat denaturation at 94 C
step 2 - primers anneal at 55 C
step 3 - primer extension using nucleotides at 72 C
exponential process
describe the PCR product analysis with an example
1) detect presence eg DNA deletion in CFTR gene with CF - has a 3 BP deletion and therefore carried faster in gel electrophoresis
2) OLA - oligonucleotide ligation assay ie allele specific mutation must be identified
how are OLA distinguished in mutant genes
allele specific oligonucleotides are designed so 3’ end base pair with variable nucleotide
ie chains won’t ligate unless perfectly base paired
what is the problem with identifying unknown genes
polymorphism - normal variations between genomes so mis be able to distinguish between pathogenic mutations and normal ones
what is sanger DNA sequencing
chain terminators which terminate the DNA at a specific base pair which shows up as a band
what does sanger DNA sequnecning allow us to do
show where the pathogenic mutation is and whether the individual is homozygous or heterozygous
what are the limitations of sanger DNA sequencing
limited to around 500 bp long can’t see full genome
not cheap or quick
exon targeted
what is a clinical exome
8000 genes = 25 Mbp which is a cheaper method
there are many variants so focus should be on specific suspected illness
(not good for large scale genome pathology, only single base changes)
what method would you sue for large scale genome pathology screening
cytogenic analysis - identify chromosome number and structure = G banding during metaphase under a microscope (resolution is limited)
what is FISH genome testing
Fluorescent in situ hybridisation
much better resolution than G banding
combines labelling of chromosomes with labelling of bases = can detect loci
as well as detection of mcirodeletion on chromosomes
give an example of a disease than can be identified using FISH
DiGeorge syndrome - 2-Mbp deletion of chromosome 22q11.2
how are centromeric probes used in FISH
probes on centromeres identify chromosomes - if a chromosome has a probe and the other doesn’t then there is a deletion
(used in interphase of chromosomes)
