dc.description.abstract |
Aim: This work sets out to use haplotype-based tagSNP selection and a systematic in
silico analysis for design of multiplex-compatible PCR primer and SAT probe sets to
capture maximum variation with minimum tests across candidate genes IGF1, IGFBP1
and IGFBP3. Additionally, the work aims to develop a number of robust, high-efficiency,
high-specificity multiplex PCR constructs for amplification of these targets and to
demonstrate the applicability of these target types to suspension array genotyping for
non-insulin-dependant diabetes mellitus association facilitation.
Methods: Haplotypes for predominantly European Caucasian populations were
constructed and tagSNP selection performed using Haploview to capture maximum
variation across candidate genes IGF1, IGFBP1 and IGFBP3. Extensive in silico analysis
was performed for design, evaluation and selection of robust high-specificity primer and
probe pairs, suitable for downstream multiplex PCR and SAT analysis. Singleplex endpoint
and real-time PCR was performed for primer pair profile determination which
informed multiplex PCR set construction and optimisation. The applicability of this
complex target type to suspension array-based genotyping was investigated using a
model probe pair using both quantum dot-encoded and fluorophore-encoded
microspheres.
Results: Haploview was used for haplotype construction and linkage disequilibriumbased
tagSNP selection across candidate genes, reducing the number of SNP targets from
292 to 32 with minimal information loss. Extensive evaluation of potential tagSNPs was
performed and 29 SNPs, representing 29 bins across target genes were designed for
multiplex analysis. Singleplex end-point and real-time PCR was performed for primer
pair profile determination which allowed four multiplex PCR sets to be constructed and
optimised for simultaneous amplification of 14, six, five and two targets. The
applicability of this complex target type (14-plex) to suspension array-based genotyping
was demonstrated using a model probe pair.
Conclusion: In silico analysis techniques have been applied for successful development
of four robust multiplex PCR sets (14-plex, 6-plex, 5-plex and 2-plex) which display
high-efficiency and target-specific amplification of tagSNPs, capturing maximum assaycompatible
variation across candidate genes IGF1, IGFBP1 and IGFBP3 for European
Caucasian populations. The applicability of these multiplex PCR constructs to suspension
array-based genotyping has been demonstrated, thus paving the way for development of
large multiplex suspension array-based genotyping assays using probes designed during
the course of this work. This work offers the potential for comprehensive association
analyses to become more accessible to the wider-scientific community by facilitating
reduced genotyping burdens which allow increased accessibility for powerful association. |
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