Leader: Chris Mattocks
Mutation screening can be split in to two fundamental
processes. A primary screen is used to detect the presence of
a sequence variation in a particular fragment. This may or may
not characterise the variation in question. A secondary screen
(usually by sequencing) is used to confirm the results of the
primary screen with respect to the particular sample in question
and may also be used to characterise mutations if this is not
a function of the primary screen. One of the most fundamental
questions regarding which methodology should be used for high
throughput mutation screening for large genes is whether to use
a direct sequencing approach or an indirect approach for the
primary screen. We have carried out extensive theoretical analysis
of this question and have identified the main factors that need
to be considered in deciding between the two methodologies. A
simplified model can be viewed here.
A more sophisticated spread sheet model that we have used extensively
is available for down
loading together with explanatory notes.
Explanatory notes: Pre
screen modelling explanatory notes.doc (41.0KB)
modeling.xls (84.0 KB)
Obviously the primary consideration with any screen
will be the sensitivity. However assuming a methodology has a
satisfactory sensitivity the main factors are: the relative ‘value’ of
the pre-screen (in terms of either time per test, or cost per
test) compared to sequencing, the number of fragments required
to be analysed to achieve a result on a particular patient and
the failure rates of the pre-screen method compared to that of
a direct sequencing strategy.
Using SPODS we have
evaluated two different methods of mutation screening applied
to the same set of fragments. In house we have set up confirmation
sensitive capillary electrophoresis (CSCE)
using a protocol originally developed and validated by the
Cancer Genome group at the Sanger Centre. This has proved to
extremely sensitive and very rapid.
We have also
evaluated a novel mass-spectrometry application produced by Sequenom.
This uses T7 promoter mediated transcription followed by MALDI-TOF
detection of base specific RNA cleavage products (MassCleave™).