7. molecular testing Flashcards
Non sense mutation
point mutation that causes a premature stop codon
Mis sense mutation
point mutation that results in a change of amino acids
silent mutation
point mutation that does not result in a change of amino acid
frameshift mutation
addition or deletion of a DNA base changes the reading frame
expansion mutation
expansions of triplet repeat sequence within coding or non coding regions may show anticipation- age of onset lower, worse severity in successive generations due to progressive increase in size of repeats thresholds for repeats eg. >40 Huntingtons
hotspot
genes with regions that show high frequency of mutation - usually functionally important areas and allow tests to be targeted at
allele heterogeneity
multiple different mutations within the same gene may give the same phenotype
locus heterogenity
mutations in different genes in a pathway may give same syndrome increases number of tests required to screen for the syndrome
germline mutation
inherited, present in every cell
acquired mutations
somatic- present in diseased tissue
common mutation in CF
delta F508
when must the whole gene be screened
when the mutation shows allelic heterogeneity eg. BRCA1
challenges facing molecular tests
hotspots allele heterogeneity different mutations within same gene may give different phenotypes locus heterogeneity
PCR
amplify specific region of interest eg. hotspot a form of in-vitro DNA replication
Post PCR analysis: size
evaluated by size by electrophoresis identify expansion mutations polymorphic micro-satellite markers to be followed for linkage analysis
post PCR analysis: presence or absence of a product
absence of a PCR product (eg. missing band) show deletion can be used to look for dystrophin deletions beware if only top bands deleted: pcr failing? beware if only one band deleted: mispriming due to SNP?
Multiplex ligation probe amplification MPLA
- normal sequencing - screens large no. of exons - test for deletions tests for quantifications
amplification refractory multiplex system (ARMS)
PCR with mutation specific primers so known mutations can be detected
pre screening by conformation changes
allelic heterogeneity- lots of regions need to be tested - expensive to sequence each one
allows for PCR to be tested for sequence change before definitive sequencing
Heteroduplex
- altered migration on electrophoresis and binding on HPLC columns
- > 90% sensitivity
- commonly used in NHS
- presence of mutant and wild type- melt and re anneal. heteroduplex migrate
Single strand conformation
- altered by base changes
- altered migration on electrophoresis
- <70%, cheap for research
- denature DNA to single strands, secdonary structure formed- reannealed mutant forms different structure to wild type
Post PCR analysis: sequencing
definitive answer!
Dideoxy sequencing (Sanger sequencing) most commonly used, expensive
pyrosequencing is new and more sensitive but can only produce sequence of fragments
all mutations should be validated by sequencing but not easy
Methylation specific PCR
used to detect imprinted or epigenetically silenced alleles
DNA is modified by bisulphite reaction which converts non-methylated cytosines to thymine
methylation specific primers can be used to test for presence or absence of PCR product
