Genomic DNA was isolated from peripheral blood by a standard salting-out procedure [65 (link)]. DNA samples were genotyped for the MAOB (rs1799836 and rs6651806), HTR2A (rs6314 and rs6313), and HTR2C (rs3813929 and rs518147) gene polymorphisms using TaqMan-based allele-specific polymerase chain reaction (PCR) on an ABI Prism 7000 Sequencing Detection System apparatus (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s procedures. Briefly, 20 ng of genomic DNA was PCR amplified in 96-well plates using a 10 μL reaction volume. The conditions of the PCR reaction were as follows: initially, 95 °C for 10 min, then 40 cycles at 92 °C for 15 s, and 60 °C for 60 s.
Abi prism 7000 sequencing detection system apparatus
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7000 Sequencing Detection System is a real-time PCR instrument designed for DNA amplification and detection. It is capable of performing quantitative and qualitative analysis of nucleic acid samples.
Automatically generated - may contain errors
3 protocols using abi prism 7000 sequencing detection system apparatus
1
Genotyping of Neurotransmitter Receptor Genes
2
Genotyping of Gastric Cancer Genes
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Genomic DNA from gastric tumour and nontumour tissues was extracted using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) and QuickGene™ DNA Tissue Kit S (Fujifilm Corporation, Tokyo, Japan) on QuickGene-810 DNA isolation system (Fujifilm) according to manufacturer’s protocol. Genomic DNA from control population was extracted from peripheral blood samples using Wizard® Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol. The DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.). Genotyping for polymorphism rs151658 (C>G) in TTK gene, and polymorphisms rs1031963 (C>T) and rs1801376 (A>G) in BUB1B gene was performed using TaqMan-based allele-specific polymerase chain reaction assays on the ABI Prism 7000 Sequencing Detection System apparatus (Applied Biosystems, Foster City, CA, USA) according to the procedure recommended by Applied Biosystems. The 10 μL reaction volume contained 100 ng of DNA. Assay IDs were: C_3181603_10, C_1237153_10, and C_3052718_1. In order to confirm the veracity of the results, the polymorphisms were re-genotyped by direct sequencing on a randomly selected smaller batch of samples.
3
GABRA2 gene variants genotyping
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Blood samples (8 ml) from alcohol-dependent patients and control subjects were drawn in a plastic syringe with 2 ml acid citrate dextrose anticoagulant. Genomic DNA was isolated from peripheral blood leukocytes according to standard procedures by a salting out method. Three SNPs located in the 5'and central region of the GABRA2 gene and in the intergenic region between GABRA2 and GABRG1 genes on chromosome 4 were analyzed.
Namely, rs567926 (3' flanking region), rs279858 (exon 5) and rs9291283 (intron 3) were genotyped using Taqman TM probe-primer combinations (Cat. No. C_7537087_10, C_2073557_1_ and C_8262290_10), available from the Applied Biosystems Assay-on-Demand TM human SNP genotyping collection (Applied Biosystems, Foster City, CA, USA).
Taqman-based allele-specific polymerase chain reactions and post-PCR fluorescence plate reads were performed according to the procedure described by Applied Biosystems, using an ABI Prism 7000 Sequencing Detection System apparatus. Briefly, 20 ng of genomic DNA was PCR amplified in 96-well plates using a 10 l reaction volume for 40 cycles at 92°C for 15 s followed by 60°C for 60 s. Allele nucleotide designation of the analyzed SNPs refers to the chromosome plus strand sequence.
Namely, rs567926 (3' flanking region), rs279858 (exon 5) and rs9291283 (intron 3) were genotyped using Taqman TM probe-primer combinations (Cat. No. C_7537087_10, C_2073557_1_ and C_8262290_10), available from the Applied Biosystems Assay-on-Demand TM human SNP genotyping collection (Applied Biosystems, Foster City, CA, USA).
Taqman-based allele-specific polymerase chain reactions and post-PCR fluorescence plate reads were performed according to the procedure described by Applied Biosystems, using an ABI Prism 7000 Sequencing Detection System apparatus. Briefly, 20 ng of genomic DNA was PCR amplified in 96-well plates using a 10 l reaction volume for 40 cycles at 92°C for 15 s followed by 60°C for 60 s. Allele nucleotide designation of the analyzed SNPs refers to the chromosome plus strand sequence.
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