May 28, 2024
Filaggrin genotyping using Taqman SNP genotyping assays
- 1Genetics Core, Edinburgh Clinical Research Facility, University of Edinburgh
Protocol Citation: Angie Fawkes, Katarzyna Hafezi, Lee Murphy 2024. Filaggrin genotyping using Taqman SNP genotyping assays. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl492mogo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 08, 2024
Last Modified: May 28, 2024
Protocol Integer ID: 97917
Keywords: Filaggrin, Taqman, Genotyping, SNP, FLG
Abstract
The FLG gene encodes the filaggrin protein which is essential for epidermal barrier formation and hydration. Mutations in the filaggrin gene are associated with a broad range of skin and allergic diseases. This protocol is used to genotype four mutations (R501X, 2282del4, R2447X and S3247X) within the FLG gene.
Materials
Applied Biosystems QuantStudio5
Taqman Genotyping Master Mix (Applied Biosystems Cat. no. 4371355)
MicroAmp Optical 384-Well Reaction Plate, 1,000 plates (Applied Biosystems Cat. no. 4343370)
MicroAmp Optical Adhesive Film, 100 films (Applied Biosystems Cat. no.4311971)
UltraPure Nuclease-free water, 500ml (Invitrogen Cat. no. 10977035)
FLG mutation-specific primers and probes:
Sequence Detection Primer (Applied Biosystems Cat. no. 4304970)
Taqman TAMRA Probe (Applied Biosystems Cat. no. 450025)
Taqman MGB Probe (Applied Biosystems Cat. no. 4316034)
| A | B | C | |
| Mutation | Primer pairs | Probes | |
| R501X | Forward: 5’ CAC TGG AGG AAG ACA AGG ATC G 3’ Reverse: 5’ CCC TCT TGG GAC GCT GAA 3’ | Wild type 5’ 6-FAM-CAC GAG ACA GCT C-MGBNFQ 3’ Mutant 5’ VIC-CAT GAG ACA GCT CC-MGBNFQ 3’ FAM=C; VIC=T | |
| S3247X | Forward: 5’ CCA GAA ACC ATC GTG GAT CTG 3’ Reverse: 5’ TGC CTG ATT GTC TGG AGC G 3’ | Wildtype: 5’ 6-FAM-CAG TCA AGG CAC GG-MGBNFQ 3’ Mutant: 5’ VIC-AGC AGT AAA GGC ACG-MGBNFQ 3’ FAM=C; VIC=A | |
| 2282del4 | Forward-1: 5’ TCC CGC CAC CAG CTC C 3’ Forward-2: 5’ CCA CTG ACA GTG AGG GAC ATT CA 3’ Reverse: 5’ GGT GGC TCT GCT GAT GGT GA 3’ | Wild type: 5’ 6-FAM-CAC AGT CAG TGT CAG GCC ATG GAC A-TAMRA 3’ Mutant: 5’ VIC-AGA CAC ACA GTG TCA GGC CAT GGA CA-TAMRA 3’ FAM=T; VIC=A | |
| R2447X | Forward-1: 5’ ACG TGG CCG GTC AGC A Forward-2: 5’ AGC ACT GGA GGA AGA CAA GGA T 3’ Reverse: 5’ CCT GAC CCT CTT GGG ACG T 3’ | Wild type 5’ 6-FAM-CAC GAG ACA GCT C-MGBNFQ 3’ Mutant 5’ VIC-CAT GAG ACA GCT CC-MGBNFQ 3’ FAM=C; VIC=T |
Primers and probes for FLG mutation screening by Taqman allelic discrimination
Prepare primers and probes
Prepare primers and probes
Primers are sent in a concentration of 20pmol/μL and volume 500μL. To prepare primers mix 500μL of primers at 20pmol/μL with 100μL of nuclease-free water.
To prepare probes mix an equal volume of probe with nuclease-free water (based on a probe concentration of 6,000 pmol).
Prepare reaction mix
Prepare reaction mix
The tables below are for one sample based on 10μL reaction volume (2μL of DNA and 8μL of reaction mix). Multiply up reaction mix by number of samples plus extra for dead-volume. Ensure a sensible amount for accurate pipetting.
(This means that for R501X and S3247X primers will be at 300nM & probes at 100nM; and for 2282del4 and R2447X - Forward and Reverse primers will be at 500nM, Inner Forward Primer 2 at 100nM & probes at 100nM).
For R501X and S3247X prepare a mastermix based on per sample volumes in the table. Prepare a separate mastermix for R501X and for S3247X:
| A | B | |
| Reagent | Volume (μL) | |
| Primer Forward | 0.03 | |
| Primer Reverse | 0.03 | |
| Probe 1 | 0.02 | |
| Probe 2 | 0.02 | |
| Taqman Genotyping Master Mix (2x) | 5 | |
| Nuclease-free water | 2.90 |
For 2282del4 and R2447X prepare a mastermix based on per sample volumes in the table.
| A | B | |
| Reagent | Volume (μL) | |
| Primer Forward | 0.05 | |
| Primer Reverse | 0.05 | |
| Probe 1 | 0.02 | |
| Probe 2 | 0.02 | |
| Inner Forward Primer 2 | 0.01 | |
| Taqman Genotyping Master Mix (2x) | 5 | |
| Nuclease-free water | 2.85 |
Prepare reaction plate
Prepare reaction plate
Add 2μL of DNA at a concentration of 10ng/μL, for each sample to be genotyped, to a 384-well plate. Ensure at least 16 wells of the 384 have no DNA added for blank controls (these wells should still have reaction mix added).
PCR amplification
PCR amplification
Create an experiment on QuantStudio5 or load an existing experiment template (EDT file) using QuantStudio Design & Analysis Software. Ensure assay file is uploaded into library. See QuantStudio user manual for details.
Load 384-well plate onto heating block ensuring plate is in correct orientation with well A1 positioned top-left.
Cycle with the following settings:
60 °C for 00:00:30
95 °C for 00:10:00
Then 40 cycles of
Denaturation: 95 °C for 00:00:15
Annealing/extension: 60 °C for 00:01:00
Then 60 °C for 00:00:30
12m 15s
Data analysis
Data analysis
Within QuantStudio Design & Analysis Software review data to ensure clear clusters are visible and that blanks are not called.
For each genotype, export data to excel and save a copy of each allelic discrimination plot as a pdf file.
The FLG genotyping is looking for rare alleles so does not form the usual three clusters. The following clusters are examples to help determine final genotypes.
Cluster plot for R501X
Cluster plot for S3247X
Cluster plot for 2282del4
Cluster plot for R2447X
Data Report
Data Report
Prepare the data summary report. Include information on scanner used, SOP, analysis software version and assays used. Detail call rates and any false call rates (blanks that gave a genotype). Return summary report with raw data (eds files), exported data (excel files) and allelic cluster plots (pdf).
Protocol references
Sandilands, A., Terron-Kwiatkowski, A., Hull, P. et al. Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema. Nat Genet 39, 650–654 (2007). https://doi.org/10.1038/ng2020.
Genotyping Analysis Module User Guide for use with QuantStudio Design and Analysis Software v2. Publication Number: MAN0018749. Revision: D.0
(Accessed 8 Apr 2024)