Transcript SNPs - Bilkent University

SNPs
Human Genome
SNP Typing
• Allele specific
hybridization
• ASO probes
usually with the
polymorphic
base in a
central position
in the probe
sequence
SNP Typing
• minisequencing reaction
or single nucleotide
primer extension where
the polymorphic base is
determined by the
addition of the ddNTP
complementary to the
base interrogated by a
DNA polymerase.
• Secondly there is the
allele-specific extension
where the DNA
polymerase amplifies
only if the primers have
a perfect match with the
template.
SNP Typing
•
•
•
Taqman: TaqMan; this assay is
based in the 5’nuclease activity
of Taq polymerase. When the
probes are intact, the quencher
interacts with the fluorophore by
FRET, quenching their
fluorescence.
one probe is complementary to
the wild-type allele and the other
to the variant allele. These
probes have different fluorescent
dyes attached to the 5’end and a
quencher attached to the 3’ end.
In the extension step, the 5’
fluorescent dye is cleaved by the
5’ nuclease activity of the Taq
polymerase, leading to an
increase influorescence of the
reporter dye. Mismatch probes
are displaced without
fragmentation.
ADAM33 Astma
• Linkage analysis performed using multipoint linkage: LOD score (MLS) of
2.94 near D20S906 and a second MLS of 2.94 at D20S482.
• 218 nuclear families, the asthma plus BHR phenotype increased the
evidence for linkage (MLS of 3.93 at D20S482, 35% excess allele sharing)
and refined the candidate region to the second peak
• The region spanned 4.28 centimorgans (cM) (from 9.08 cM to 13.36 cM,
Fig. 1b) within markers D20S842 and D20S849. (BAC) clones were
sequenced to facilitate gene identification
Identification of 40 genes
• A combination of public data mining,
complementary DNA (cDNA) library
screening, direct cDNA selection and
polymerase chain reaction with reverse
transcription (RT–PCR) was used to
characterize 40 genes
Association Studies
• Unrelated affected offspring from families
showing evidence for linkage were selected as
cases.
• Caucasian controls were collected in both the
US and the UK in order to match the country of
origin with the cases.
• On average, 130 IBD-affected individuals and
217 ‘hyper-normal’ controls were compared for
allele and genotype frequencies.
Adam33
• Analyses of 135 nucleotide polymorphisms
(SNPs) in 23 genes spanning the 90%
confidence interval revealed that the
ADAM33 region showed the most
significant association signal in the linkage
region.
• 130 kilobases (kb) to the left of ADAM33
and up to 40 kb to the right.
Adam33
• Within this ,185-kb linkage disequilibrium
interval, 24 SNPs in three genes were
significant in either the combined or
separate US/UK analyses.The majority of
these SNPs (14) were in ADAM33.
Linkage Disequilibrium
Gene Structure and SNPs