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Iplex gold reagent kit

Manufactured by Agena

The IPLEX Gold Reagent kit is a laboratory equipment product that provides a core function for specific applications. The kit contains essential components required for designated procedures. No further details or interpretation are available.

Automatically generated - may contain errors

3 protocols using iplex gold reagent kit

1

Genotyping of Lipid-Associated Genetic Variants

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Genotyping of PNPLA3 rs738409, NCAN rs2228603, GCKR rs780094, LPRLAL1 rs12137855 and 59 ancestry informative SNPs was performed using single base extension (Sequenom, San Diego, CA). Assay Design Suite, V2.0 (Agena Bioscience, San Diego, CA) was used to design amplification and extension primers. PCR, neutralization of unincorporated dNTPs and single base extension were performed using the iPLEX Gold Reagent kit (Agena Bioscience, San Diego, CA). Data were generated with Spectro ACQUIRE software version 3.0 and analyzed using Typer software, version 4.0.22 (Sequenom). Genotyping was performed at the Johns Hopkins Institute for Clinical & Translational Research (ICTR) laboratory.
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2

Validating De Novo Mutations via Sequencing

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All putative de novo mutations were tested by both Sanger sequencing and the MassARRAY system (Agena Bioscience, San Diego, CA). For assays with the MassARRAY system, PCR amplification and single base extension primers for each mutation were designed using Assay Design Suite software (Agena Bioscience), and PCR amplification and single base extension reactions were performed using reagents supplied in the iPLEX Gold reagent kit (Agena Bioscience). To evaluate background noise, PCR reactions containing no template DNA were also performed. MassARRAY data were analyzed using a Typer Analyzer (Agena Bioscience), which facilitates visualization of the raw spectra and determines the degree of confidence for wild-type and mutant peak signals. A true mutant was defined as having a peak probability of at least 0.95. For assays with Sanger sequencing, the same PCR amplification primers for MassARRAY assays were used. The Sanger sequencing chromatograms were visualized using Chromas V2.6.6 (https://technelysium.com.au/wp/). The presence or absence of a mutation was determined manually by whether there is a mutant peak at the target site.
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3

Multiplexed Genotyping for MS Risk Variants

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Sequenom platform Iplex Gold Reagent Kit multiplexed genotyping assays were designed in ~25 SNP pools using the Assay Design Suite (Agena Biosciences) for 186 of 200 putative non-MHC MS risk variants.22 Linked discovery SNPs or tagging SNPs were substituted for effect SNPs refractory to multiplex design. Samples were spotted to Sequenom SpectroChip(s) and scanned with MALDI-TOFMS. Non-template negative controls, sample duplicates and control DNAs were included. HLA-DRB1*15:01 alleles were determined via TaqMan genotyping of SNP rs9271366 (assay ID: C__33416976_20). HLA-A*02 alleles were determined via TaqMan genotyping of the SNP rs2975033 (assay ID: C__15962692_20). Each SNP assay was reviewed for the genotype call quality based on PCR/Extension yield, proximity to cluster, and raw mass spectra.
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