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Iplex gold genotyping reagent set

Manufactured by Agena
Sourced in United States

The IPLEX Gold Genotyping reagent set is a laboratory equipment product designed for genetic analysis. It provides the necessary reagents and components to perform genotyping experiments. The core function of this product is to enable the detection and identification of specific genetic variants or polymorphisms within a sample.

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5 protocols using iplex gold genotyping reagent set

1

Multiplex Genotyping Protocol with MassArray

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Assays were designed with Assay Designer 4.0 (Agena) and analysis performed using iPlex Gold Genotyping Reagent Set (Agena, San Diego, CA) according to manufacturer’s instructions. Target loci were amplified within the samples by multiplex PCR in 1X PCR buffer containing 3.5 mM MgCl2, 25 mM dNTPs, 500 nM each of forward and reverse amplification primer within the multiplex pool and 2.5 U HotStar Taq. dNTPs and primers were removed by incubation with 0.5 U shrimp alkaline phosphotase (SAP) at 37 °C for 40 minutes. SAP was inactivated by incubation at 87 °C for 5 minutes. Single base extension was carried out in 0.2X iPLEX buffer plus, 1X termination mix (containing mass modified termination nucleotides), 1X iPLEX enzyme and primers at 0.84 μM, 1.04 μM and 1.25 μM as appropriate to the relative mass of each primer. Following thermocycling, clean resin and water was added to the MassExtend reaction products. Samples were incubated in clean resin at room temperature with mixing for 5 minutes and centrifuged at 3200 × g for 5 minutes.
Samples were then dispensed to a SpectraChip using the MassArray Nanodispenser according to manufacturer’s instructions. Spectra chips were loaded into the MassArray analyzer and spectra acquired for each sample. Genotype calls were made using Typer 4.0 (Agena) by mass identification of extended primer peaks.
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2

Multiplex Genotyping Protocol with MassArray

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Assays were designed with Assay Designer 4.0 (Agena) and analysis performed using iPlex Gold Genotyping Reagent Set (Agena, San Diego, CA) according to manufacturer’s instructions. Target loci were amplified within the samples by multiplex PCR in 1X PCR buffer containing 3.5 mM MgCl2, 25 mM dNTPs, 500 nM each of forward and reverse amplification primer within the multiplex pool and 2.5 U HotStar Taq. dNTPs and primers were removed by incubation with 0.5 U shrimp alkaline phosphotase (SAP) at 37 °C for 40 minutes. SAP was inactivated by incubation at 87 °C for 5 minutes. Single base extension was carried out in 0.2X iPLEX buffer plus, 1X termination mix (containing mass modified termination nucleotides), 1X iPLEX enzyme and primers at 0.84 μM, 1.04 μM and 1.25 μM as appropriate to the relative mass of each primer. Following thermocycling, clean resin and water was added to the MassExtend reaction products. Samples were incubated in clean resin at room temperature with mixing for 5 minutes and centrifuged at 3200 × g for 5 minutes.
Samples were then dispensed to a SpectraChip using the MassArray Nanodispenser according to manufacturer’s instructions. Spectra chips were loaded into the MassArray analyzer and spectra acquired for each sample. Genotype calls were made using Typer 4.0 (Agena) by mass identification of extended primer peaks.
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3

Targeted Genotyping of Retinal Variants

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Candidate variants associated with a missense change in a RetNet gene were selected for genotyping. Assays were designed using the MassARRAY Assay Design included in the package Typer 4.0 (Agena Bioscience, San Diego, CA) according to manufacturer’s instructions. A multiplex consisting of seven SNPs in six different genes (CHD3, CNGB1, DTHD1, RBP3, USH1C and CDH23) was selected (Supplementary Table 1), genotyping was performed on all available samples using the Agena Biosciences iPLEX Gold Genotyping reagent set and products were typed with the MassARRAY System with Nanodispenser RS1000 (Agena Bioscience).
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4

Validating Novel HR Variants in Cats

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The two HR frameshift variants, including an exon 3 c.1255_1256dupGT, and the exon 18 c.3389insGACA, were identified by the WGS analyses. These variants were validated in the WGS cat by Sanger sequencing (Supplementary Table S1) A mass spectroscopy assay was designed as previously described [32 (link)] to genotype the identified variants in pedigree A (Supplementary Figure S1) and the additional cats, using the Agena Bioscience iPLEX Gold Genotyping reagent set (Agena Bioscience Inc., San Diego, CA, USA) (Supplementary Table S2). Products were genotyped with the MassARRAY System with Nanodispenser RS1000 (Agena Bioscience Inc., San Diego, CA, USA).
Not all ascertained cats with similar hair coats had the WGS identified variants, therefore the coding regions of HR were Sanger sequenced in each additional founder cat (Supplementary Table S1). PCR and thermocycling conditions were conducted as previously described [43 (link)]. The variants for the cats in the pedigree B (Supplementary Figure S2) were also genotyped by Sanger sequencing.
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5

Genetic Variant Screening in Cats

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Archival DNA samples from 510 non-BSH cats of 29 different non-BSH breeds and random bred cats were screened using mass spectrometry (MassARRAY, Agena Bioscience, San Diego, CA). An assay was designed to type the identified variant (Table 1), and genotyping was performed on the samples using the Agena Bioscience iPLEX Gold Genotyping reagent set. Products were typed with the MassARRAY System with Nanodispenser RS1000 (Agena).
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