SUPPLEMENTARY METHODS
SNaPShot PCR assay for BRAF mutations
The wild type nucleotide sequence at BRAF codon 600 is GTG which corresponds to
nucleotides 1798, 1799 and 1800 of the BRAF nucleotide sequence. The most common
mutation is a T>A at nucleotide 1799 resulting in the amino acid changing from valine to
glutamic acid (V600E). However, base changes at nucleotides 1798 and 1800 can also result
in mutations (V600D, V600K and V600R).
All these variants can be readily detected by Sanger sequencing of a PCR amplicon spanning
codon 600. However, the sensitivity of Sanger sequencing is about 15–20%. In order to
detect lower levels of mutations we implemented the SNaPshot technique which has a
sensitivity of about 5%. Basically SNaPshot is minisequencing where a primer that
terminates at the mutation site is extended using a mixture of all four fluorescently labelled
dideoxynucleotides. Only one nucleotide is incorporated and this is determined by the
template. This test is set up to detect mutations at nucleotides 1798, 1799 and 1800. In order
to capture these in a single multiplex assay, the detection primers were made to different
lengths by adding non-specific sequences at the 5’ end and thus could be easily distinguished
by their size on a capillary sequencer. The actual nucleotide incorporated is determined by its
colour. The principle is illustrated below.
The interpretation is assisted by the table below.
Mutation
1799
1800
1798
W/T
T
C
G
V600R
G
C
A
V600K
A
C
A
V600E
A
C
G
V600E2
A
T
G
V600M
T
C
A
V600G
G
C
G
V600D
A
A
G
The colours of the dideoxynucleotides used are
Dideoxy G
Blue
Dideoxy A
Green
Dideoxy C
Black
Dideoxy T
Red