7. molecular testing

Question 1

Non sense mutation

Answer 1

point mutation that causes a premature stop codon

Question 2

Mis sense mutation

Answer 2

point mutation that results in a change of amino acids

Question 3

silent mutation

Answer 3

point mutation that does not result in a change of amino acid

Question 4

frameshift mutation

Answer 4

addition or deletion of a DNA base changes the reading frame

Question 5

expansion mutation

Answer 5

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

Question 6

hotspot

Answer 6

genes with regions that show high frequency of mutation - usually functionally important areas and allow tests to be targeted at

Question 7

allele heterogeneity

Answer 7

multiple different mutations within the same gene may give the same phenotype

Question 8

locus heterogenity

Answer 8

mutations in different genes in a pathway may give same syndrome increases number of tests required to screen for the syndrome

Question 9

germline mutation

Answer 9

inherited, present in every cell

Question 10

acquired mutations

Answer 10

somatic- present in diseased tissue

Question 11

common mutation in CF

Answer 11

delta F508

Question 12

when must the whole gene be screened

Answer 12

when the mutation shows allelic heterogeneity eg. BRCA1

Question 13

challenges facing molecular tests

Answer 13

hotspots allele heterogeneity different mutations within same gene may give different phenotypes locus heterogeneity

Question 14

PCR

Answer 14

amplify specific region of interest eg. hotspot a form of in-vitro DNA replication

Question 15

Post PCR analysis: size

Answer 15

evaluated by size by electrophoresis identify expansion mutations polymorphic micro-satellite markers to be followed for linkage analysis

Question 16

post PCR analysis: presence or absence of a product

Answer 16

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?

Question 17

Multiplex ligation probe amplification MPLA

Answer 17

• normal sequencing - screens large no. of exons - test for deletions tests for quantifications

Question 18

amplification refractory multiplex system (ARMS)

Answer 18

PCR with mutation specific primers so known mutations can be detected

Question 19

pre screening by conformation changes

Answer 19

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

Question 20

Post PCR analysis: sequencing

Answer 20

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

Question 21

Methylation specific PCR

Answer 21

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