MRC Path - Mutation Detection Methods
Basis of the test:
Two oligonucleotide probes are hybridised to adjacent
sequences of PCR amplified target DNA with the join situated at
the position of a known mutation site. DNA ligase covalently joins
the two oligonucleotide probes only if they are perfectly hybridised
which will only occur if no mutation is present. Initial studies
tagged one probe with biotin and the other with a reporter molecule
such as digoxygenin. On transfer to streptavidin coated microtitre
plates washing will remove the reporter labelled probe unless
ligation has occurred. Signal detection will therefore only occur
if the mutation is not present (or visa versa depending
on the design of the probe). This technology was improved when
Tobe et al developed a method of typing two alleles in a single
microtitre well by marking each of the allele specific primers
with different enzyme reporter molecules so that two different
colours can be produced.
The fact that this technique does not involve centrifugation
or electrophoresis makes it amenable to large scale automated
testing.
Recent advances to the technique have replaced the
biotin with mobility modifying tails and digoxygenin with fluorescent
tags (FAM, HEX or TET) with the ligation products being electrophoresed
and analysed on an automated sequencer. These modifications have
allowed the number of known point mutation sites which can be
screened at any one time to be increased and Applied Biosystems
now have kits capable of screening for 31 known CF mutations in
a single reaction tube and single lane of an electrophoresis run.
The Applied Biosystems CF Assay
A cocktail of fifteen pairs of primers is used to
amplify regions of the CFTR gene where common mutations are located.
Each amplified segment (or amplicon) is probed with three oligonucleotide
probes. The common probe hybridises to the amplicon at a sequence
that is present in both normal and mutant alleles and is labeled
with one of three fluorescent tags.
The mutant and normal probes are allelic and can
be distinguished by their length since they have varying numbers
of mobility modifying tails attached. These tails were initially
non-complementary Poly A or Poly C but are now composed of Pentaethylene
oxide (PEO) units.
At any one locus, if the mutation is not present
only the normal and common probe will form a ligation product,
if a homozygous mutation is present only the mutant and common
probes will ligate and in heterozygous samples both normal and
mutant probes will ligate to the common probe in equal amounts
but giving products of different mobility.
The rTth DNA ligase facilitates ligation by catalysing
the formation of a phosphodiester bond between the 5' phosphate
of the common probe and the 3' hydroxyl of either the normal or
mutant probe. This reaction will ONLY occur when both probes are
hybridised and perfectly matched to the complementary target amplicon.
Following ligation, the product for each amplicon
has a unique combination of electrophoretic mobility and fluorescence
which allows identification of the sample phenotype by size separation
of the ligation product in each of the three colours (the CF kit
is designed to have 10-12 blue, 10-12 green and 9-11 yellow peaks).
A red labelled internal size standard allows Genescan analysis
software to size products precisely, label fragment data and display
fragments as labelled peaks in plot displays.
The results must then be evaluated to determine accuracy.
Validation should ensure all size standard peaks are present and
correct, count peaks in both the normal and negative control lanes
and determine the validity of each labelled peak in the test sample
lanes.
Benefits:
Useful middle step between the Zeneca CF4 or 12 mutation
kits and a full rare mutation screening which is often laborious
and time consuming. Results are available quickly.
Suitable to mutation detection in any gene where
a number of known mutations or common polymorphisms occur - has
been used for screening in a number of genes e.g. the N-acrylamine-acetyltransferase
(NAT2) gene (Bigler et al) and human platelet antigen-1 (HPA-1)
(Zotz et al).
Problems:
Expensive!!
Requires good quality samples (correct blood : EDTA
ratio)
Many fragments fall outside the accepted peak height
range and must therefore be re-examined
Some polymorphisms not detected by the kit can affect
the outcome of the assay e.g. P1290P polymorphism in exon 20 causes
the amplicon used to detect W1282X to completely drop out with
the potential for false results. See table for other polymorphisms
which may have an effect:-
Table from Applied Biosytems Cystic Fibrosis Assay Protocol
| Locus of Detectable Allele | Interfering Polymorphism(s) | Possibility of Diminished OLA Product for... |
|---|---|---|
| 507 | 1648A to G 1650C to G | I507/deltaF508 and deltaI507 |
| 507 | 1651A to G | deltaI507/deltaF508 |
| 508 | 1648A to G 1650C to G | deltaI507/F508 and deltaF508 |
| 508 | 1651A to G | deltaF508 |
| 508 | 1566T to G | No diminished OLA signal at F508 locus Note F508C will not genotype as a separate allele, but will give the normal F508 genotype |
| 1282 3905 | 4002A to G | W1282, X1282, 3905 and 3905delT |
| 621+1 | 621+3A to G | 621+1G and 621+1T |
| 1162 | 3617G to T | R1162 and X1162 |
When a compound heterozygote occurs within the hotspot
region of exon 11 the corresponding normal allele for each mutation
may not be detected and consequently mutations of this region
must be re-examined.
Other problems with interpretation include dye bleed
through because of an incorrect matrix and raised base lines which
can interfere with peaks
Time required on the automated sequencer may make
the test unsuitable for routine use for all CF samples
References:
Bigler J, Chen C and Potter JD. Determination of
human NAT2 acetylator genotype by oligonucleotide ligation assay.
Biotechniques. 1997, 22 (4):682-4
Tobe VO, Taylor SL, Nickerson DA. Single-well genotyping
of diallelic sequence variations by a two- color ELISA-based oligoligation
assay. Nuc Acid Res. 1996, 24 (19): 3728-32
Zotz RB, Giers G, Maruhn-Debowski B and Scharf RE.
Genetic typing of human platelet antigen 1 (HPA-1) by oligoligation
assay in a specific and reliable semi-automated system. Br J Haematol.
1997, 96 (1): 198-203
Eggerding FA, Iovannisci DM, Brinson E, Grossman
P and Winn-Deen ES. Fluorescence-based oligonucleotide ligation
assay for cystic fibrosis trnsmembrane conductance regulator gene
mutations. Hum Mutation. 1995, 5 (2):153-65
Grossman PD, Bloch W, Brinson E, Chang CC, Eggerding
FA, Fung S, Iovannisci DA, Woo S and Win-Deen ES. High-density
multiplex detection of nucleic acid sequences: oligonucleotide
ligation assay and sequence coded separation. Nuc Acid Res. 1994,
22 (21): 4527-34
Applied Biosystems web page :- http://www.appliedbiosystems.com/products/productdetail.cfm?id=118
Applied Biosystems Cystic Fibrosis Assay - Protocol