Development of sybr green rt qpcr to confirm small snp array aberrations
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Development of SYBR Green RT-qPCR to confirm small SNP array aberrations. Carolyn Dunn , Annabel Whibley, Lionel Willatt and Ingrid Simonic Cambridge. Overview of Array Results - 2007. 134 SNP Arrays - dev delay - congenital abnormalities.

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Development of sybr green rt qpcr to confirm small snp array aberrations

Development of SYBR Green RT-qPCR to confirm small SNP array aberrations

Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic

Cambridge


Overview of array results 2007
Overview of Array Results - 2007 aberrations

134 SNP Arrays - dev delay - congenital abnormalities

60% Normal Array Result

40% ? Del/dup Array Result

Unsuitable for FISH: del <150kb or dup <1.5Mb (18%)

FISH confirmatory studies (22%)


Options for confirmatory studies
Options for Confirmatory Studies aberrations

  • A second type of array

    • Re-analysis of whole genome

    • High set-up and running costs

  • MLPA

    • Able to multiplex

    • Cost of probe expensive for single family follow-up studies

  • RT-qPCR

    • Fluorescent Probes

    • SYBR Green

      • Low cost


Sybr green rt qpcr
SYBR Green RT-qPCR aberrations

Principles

  • Denaturation of DNA to produce ssDNA

  • Thermal Cycling:

    • Primers anneal to and extend target sequence

  • SYBR Green I binds to dsDNA and emits a fluorescent signal

  • As PCR amplification proceeds, (causing the amount of dsDNA to increase), the fluorescence signal increases proportionately

ssDNA

3’

5’

3’

5’

N.B. SYBR Green I binds all dsDNA (including primer-dimers and non-specific reaction products) – essential that primers are specific to target sequence


Sybr green rt qpcr1
SYBR Green RT-qPCR aberrations

Strategy

  • Select target gene in UCSC/Ensembl

  • Export and repeat mask sequence

  • Primer design – Primer3

  • SNP check (Manchester Diagnostic SNPCheck) and BLAST primer sequences

  • PCR reaction efficiency (90–110%) and precision (Rsq value >0.985)


Overview of primer validations
Overview of Primer Validations aberrations

24 sets of primers

2 taken from RTPrimerDB

22 designed using Primer3

2 Failed QC: reaction efficiency <90 or >110% or Rsq < 0.985

Passed QC

20 Passed QC

Re-design Primers


Proof of principle study
Proof-of-principle Study aberrations

  • Is this approach reliable and robust to use diagnostically?

  • Which real time PCR machine to use?

    ABI 7900 versus Rotor-Gene 65H0

    • Ease of use, cost, consumables

    • Plates versus tubes on a rotor

  • 6 cases (5 duplications and 1 deletion)

    • Abnormal karyotype (4) or array result (2)

    • Confirmed by FISH


Set up and analysis
Set-up and Analysis aberrations

  • 4 controls

  • GAPDH used as the reference gene

  • All reactions in triplicate - SD <0.18

  • Each experiment replicated

  • Analysed using ∆∆Ct method and primer efficiency-corrected

  • Expected relative copy number

    • Normal: 1.0 (0.85-1.15)

    • Deletion: 0.5 (0.35-0.65)

    • Duplication: 1.5 (1.35-1.65)

    • Equivocal: 0.65-0.85 and 1.15-1.35



Snp array follow up data i
SNP Array Follow-up Data (I) aberrations

  • 6 SNP array abnormalities followed-up by qPCR to date (5 patients)

    • 1 was not confirmed – within the ‘normal range’

  • A high number of “calls” on the array analysis

  • One of the two array analysis packages highlighted this as an abnormality

Summary of data from 2 qPCR experiments


Snp array follow up data ii
SNP Array Follow-up Data (II) aberrations

  • 6 SNP array abnormalities followed up to date (5 patients)

    • 1 was not confirmed - same CN as controls

    • 1 borderline equivocal/duplication result

      • primer pair failed QC

      • Re-design primers and repeat

Summary of data from 2 qPCR experiments


Snp array follow up data iii
SNP Array Follow-up Data (III) aberrations

  • 6 SNP array abnormalities followed up to date (5 patients)

    • 1 was not confirmed - same CN as controls

    • 1 borderline equivocal/duplication result

      • primer pair failed QC

      • repeat with second set of primers

    • 4 confirmed (2 dels and 2 dups)


Snp array follow up data iv
SNP Array Follow-up Data (IV) aberrations

  • 300kb deletion (no suitable FISH clone)

  • 110kb deletion

  • 42kb duplication

Summary of data from 2 qPCR experiments


Snp array follow up data vi
SNP Array Follow-up Data (VI) aberrations

  • 660kb ?dupXq27.1 (includes SOX3 gene)

SOX3

X ?dupX


Summary
Summary aberrations

  • Costs higher than first predicted as primer re-design required for some cases

    • Equivocal result

    • Primers that fail QC step

  • A copy number of 4 or greater may not be accurately detected

  • A promising option for verifying small array deletions or duplications


  • Acknowledgments
    Acknowledgments aberrations

    • Dr Lucy Raymond (Clinical Genetics, Addenbrooke’s Hospital)

    • Dr Martin Curran (Head of Molecular Diagnostic Microbiology Section, Addenbrooke’s Hospital)


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