Company
Website: www.biotage.com
Pyrosequencing™ technology
is a real time sequencing method for the analysis of short to medium
length DNA sequences. Four enzymes and specific substrates are
used to produce light whenever a nucleotide forms a base pair with
the complementary base in a DNA template strand. Biotinylated PCR
products are converted to single stranded templates onto which
a sequencing primer is annealed. Analysis begins as the enzymes
and substrates are dispensed into the reaction, nucleotides are
dispensed sequentially and a light signal is detected and the base
registered. If the added nucleotide is not complementary to the
next base in the template then no light is generated. Genotypes
can be determined and quantified using the PSQ™ 96MA System
and the Allele Frequency Quantification function of the SNP software.
The following assays are currently being developed and evaluated
at NGRL (Wessex) click on relevant links to see more details:
1)
Detection of Factor V Leiden (G1691A), Factor II (G20210A ) & MTHFR
(C677T)
90 anonymised DNA samples that had been previously genotyped using
restriction enzyme digestion were re-tested using a multiplex Pyrosequencing
assay (in collaboration with Dr L Lavender, Molecular Pathology
Unit, Southampton).
2) Detection of mitochondrial mutations and estimation of mutation
load
50
anonymised DNA samples were analysed using assays designed by
NGRL (Wessex) for G3460A
(LHON), G11778A (LHON), T14484C (LHON),
A3243G (MELAS), A8344G (MERRF) & T8993G (NARP/Leighs). The
DNA samples had been genotyped previously and the level of heteroplasmy
had been estimated using (fluorescent) PCR and restriction digestion
(in collaboration with Dr A Seller, Oxford Medical Genetics Laboratory)
White
HE, Durston VJ, Seller A, Fratter C, Harvey JF, Cross NCP. Accurate
detection and quantitation of heteroplasmic mitochondrial point
mutations by Pyrosequencing. Genetic Testing. 2005;9:190-9 9
Technology Assessment Report
3) Analysis of methylation for diagnosis of Prader Willi and
Angelmans Syndrome
To determine the presence and extent of methylation at CpG
sites in Prader Willi and Angelmans syndrome, bisulfite-treated
genomic
DNA is amplified from the SNRPN gene by PCR and the C in the
CpG sites are analyzed as artificial C/T polymorphisms. Using
Pyrosequencing, the polymorphisms can be analysed and presented
with sequence context information so that methylated and non-methylated
positions can be accurately analysed. Multiple, closely spaced
CpG positions can be analyzed in one reaction. 180 anonymised
DNA samples from PW, AS and control samples are currently be
analysed in a blinded fashion.
White HE, Durston VJ, Harvey JF, Cross NCP. Quantitative
analysis of SNRPN gene methylation by Pyrosequencing as a diagnostic
test for Prader Willi Syndrome and Angelman Syndrome. Clin Chem.
March 2006; doi:10.1373/clinchem.2005.065086
Technology Assessment Report
4) Detection of trisomy 13,18 and 21 and sex aneuploidy for prenatal
diagnosis
We
have been collaborating with Prof Antonarakis at the University
of Geneva to evaluate assays that detect aneuploidy by paralogous
gene quantification 1. Sequence similarites are exploited
to co-amplify paralogue sequences located on different chromosomes.
Single nucleotide
differences between the paralogue sequences are then quantified
using Pyrosequencing technology and the ratio of the SNPs reflects
the relatively frequency of the chromosomes tested. We have been
evaluating the assay for detection of trisomy 21 and have found
the method to be reliable and robust. Assays for trisomy 13 and
18 were, however, more problematic and we have been unable to perform
reliable and
robust assays for chromosomes 13 and 18. A paper discussing our
findings is currently in preparation.
1Samuel
Deutsch, Urmila Choudhury, Giuseppe Merla, Cedric Howald, Anna
Sylvan, Stylianos E Antonarakis (2004) Detection of aneuploidies
by Paralogous Sequence Quantification. J Med Genet; 41:908-915
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