SNPs - PowerPoint PPT Presentation

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SNPs

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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 ... – PowerPoint PPT presentation

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Title: SNPs


1
SNPs
  • Human Genome

2
SNP Typing
  • Allele specific hybridization
  • ASO probes usually with the polymorphic base in a
    central position in the probe sequence

3
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.

4
SNP Typing
  • Taqman TaqMan this assay is based in the
    5nuclease 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 5end 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.

5
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

6
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 (RTPCR) was
    used to characterize 40 genes

7
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.

8
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.

9
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.

10
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11
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12
Linkage Disequilibrium
13
Gene Structure and SNPs
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