overview of genomic technologies in clinical diagnosis Flashcards
list the genomic technologies
PCR
fragment analysis
Sanger sequencing
fluorescence in situ hybridisation
array = comparative genomic hybridisation
multiplex ligation-dependent probe amplification
next generation sequencing
describe PCR
- important for many DNA applications
- used to amplify specific region of DNA
- primers flank region you want to amplify
- each cycle doubles amount of DNA copies of your target sequence
- amplify enough DNA molecules so we have sufficient material for downstream applications
describe fragment analysis
pcr based assay
pcr followed by capillary electrophoresis
sizing the pcr product
can be used to detect repeat expansions or other small size changes
repeat expansion diseases I
- Huntington’s disease = neurodegenerative disorder
- caused by CAG repeat expansion in the HTT gene
- normal <27 copies. intermediate 27-35 copies
pathogenic >35 copies - expanded protein is toxic and accumulates in neurons causing cell death
diagnosed with fragment analysis
Sanger sequencing
- cycle sequencing based on same principles as PCR
each of 4 DNA nucleotides has different dye so we can determine nucleotide sequence
up to 800bp of sequence per reaction, good for sequencing single exons of genes
slow, low throughput and costly to perform large numbers of samples
Sanger sequencing 2
detection of mutation in family by use of Sanger sequencing
R1042G mutation in gene c3 segregates with affected individuals
mutations cause disease cutaneous vasculitis
describe FISH
fluorescence in situ hybridisation
- detect large chromosomal abnormalities
- extra chromosomes
- large deleted segments
- translocations
FISH 2
- design fluorescent probe to chromosomal region of interest
- denature probe and target DNA
- mix probe and target DNA
- probe binds to target
- target fluoresces
array CGH
array comparative genomic hybridisation
for detection of sub-microscopic chromosomal abnormalities
patient DNA labelled green
control DNA labelled Red
patient array comparative genomic hybridisation profile
increased green signal over chromosomal segment in patient dna
indicates gain in patient sample not present in parents
describe MLPA
multiplex ligation dependent probe amplification = variation of PCR that permits amplification of multiple targets
each probe consists of 2 oligonucleotides which recognise adjacent target sites on DNA
we use MLPA to detect abnormal copy numbers at specific chromosomal locations
MLPA can detect sub microscopic gene deletions
MLPA 2
- one probe oligonucleotide contains sequence recognised by forward primer, other contains sequence recognised by reverse primer
- only when both probe oligonucleotides are hybridised to their retrospective targets, they can be ligated into complete probe
MPLA 3
perform fragment analysis of MLPA product
important use of MLPA is to determine relative ploidy
probes may be designed to target various regions of chromosome of a human cells
signal strengths of probes are compared with those obtained from reference DNA sample, known to have two copies of chromosomes
next generation sequencing
end to sequential testing
wider range of tests in shorter time for less money
current strategy:
- enriching to sequence only known disease genes relevant to phenotype
- panels expandable to include new genes as they are published
- pathogenic variants confirmed by Sanger sequencing
exome sequencing
- 21000 genes in human genome
- gene protein coding axons or exome represent 1-2% of genome
- 80% pathogenic mutations are protein coding
- costs £1000 for genome, 200-300 for exome
- target enrichment
- capture target regions of interest with baits
- potential to capture several Mb genomic regions (30-60)
next gen sequencing
has replaced Sanger sequencing for almost all sequencing tests in the lab
