genomic technologies in clinical diagnostics

Question 1

what is PCR

Answer 1

needed for DNA applications
amplifies specific regions of DNA
each cycle = x2 DNA
3 step process = repeated 30-40x

Question 2

describe fragment analysis

Answer 2

PCR based assay
PCR followed by capillary electrophoresis
size of the PCR product = used to detect repeat expansions or other small changes

Question 3

in reference to fragment analysis, give example of repeat expansion disease

Answer 3

Huntingtons disease
- caused by CAG repeat expansion in HTT gene
if there are more than 35 copies = expanded protein is toxic and accumulates in neurons = cell death

can be diagnosed with fragment analysis

Question 4

describe Sanger sequencing

Answer 4

cycle sequencing = same as PCR

each of the 4 DNA nucleotides have a different dye
up to 800bp of sequence per reaction
can detect mutation

BUT

slow, costly and low-throughput to perform large number of samples

Question 5

describe FISH = fluorescence in situ hybridisation

Answer 5

detect large chromosomal abnormalities e.g extra chromosomes, translocations

require fluorescent probe for region of interest
denature probe and target DNA
probe binds to target
target fluoresces

can do spectral karyotyping or target specific FISH

Question 6

describe array CGH = array comparative genomic hybridisation

Answer 6

detects sub-microscopic chromosomal abnormalaties
patient DNA = green
control DNA = red

compare green with red

Question 7

describe MLPA = multiplex ligation-dependent probe amplification

Answer 7

variation of PCR that allows amplification of multiple targets

3 main steps = hybridisation, ligation, amplification

each probe = 2 oligonucleotides that recognise adjacent target sites on DNA

used to detect abnormal at specific chromosomal locations

can detect sub-microscopic gene deletions

one probe detected by forward primer and other by reverse primer
once both probes are hybridised to their targets = ligated into complete probe

perform fragment analysis of MLPA product

use of MLPA = determine how many chromosome copies at a specific location

Question 8

describe next generation sequencing

Answer 8

wide range of tests in short time and cheap

disease panels = enrich sequence only known disease genes relevant to phenotype

Question 9

describe exome sequencing

Answer 9

21,000 genes in human genome
more efficient to only sequence parts were interested in

target enrichment = capture regions of interest with baits

Question 10

what is NHS diagnostic laboratory

Answer 10

helps consultants reach genetic diagnosis for individuals and families help guide treatment and management