Total genomic DNA was extracted from the longissimus muscle using the LaboPass TM Tissue Mini kit (Cosmo Genetech, Seoul, Korea). Two polymorphic SNPs of SREBPs and six polymorphic SNPs of the FABP4 gene in GenBank were genotyped according to Oh et al [27 (link)]. Primers for amplifications and extensions were designed for the single-base extension (Ed- this acronym is not used anywhere in the paper) for genotyping polymorphic sites [33] using forward, reverse, and extension primer sequences [27 (link)]. Reactions of the primer extension were performed using the SNaPshotddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA, USA). One unit of shrimp alkaline phosphatase was added to the reaction mixture, which was then incubated for 1 h at 37°C, followed by 15 min at 72°C for enzyme inactivation, to clean the primer extension reaction. DNA samples containing extension products and the Gene-scan 120 LIZ size standard solution were added to Hi-Di formamide (Applied Biosystems, USA) in accordance with the manufacturer’s recommendations. The mixture was incubated for 5 min at 95°C, followed by 5 min on ice, after which electrophoresis was conducted using the ABI PRISM 3130XL GeneticAnalyzer. The analysis was made using GeneMapper v4.0 (Applied Biosystems, USA).
Snapshot ddntp primer extension kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SNaPshot ddNTP primer extension kit is a molecular biology tool used for targeted DNA sequencing. It employs a single-base extension method to detect sequence variations by incorporating fluorescently labeled dideoxynucleotides (ddNTPs) at specific target sites. The kit provides the necessary reagents and components to perform this sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hi di formamide, by Thermo Fisher Scientific (4 mentions)
Genemapper v4, by Thermo Fisher Scientific (2 mentions)
Genescan 120 liz size standard, by Thermo Fisher Scientific (2 mentions)
Abi 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Amfisure pcr master mix, by GenDEPOT (1 mentions)
Quickgene dna whole blood kit s, by Fujifilm (1 mentions)
Puregene blood core kit, by Qiagen (1 mentions)
Genescan 120 liz standard, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130 avant genetic analyser, by Thermo Fisher Scientific (1 mentions)
8 protocols using snapshot ddntp primer extension kit
1
Genotyping SNPs in Muscle Tissue
2
Genetic Markers in Asthmatic Patients
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Blood samples were collected from 89 asthmatic patients comprising AERD and ATA before the ASA-BPT test. Genomic DNA was isolated from the peripheral blood using the Puregene DNA purification kit (Gentra, Minneapolis, MN, USA), according to the manufacturer’s instructions.
High-resolution typing for HLA-DBP1*0301 was performed as previously described, using the amplified gene product of locus specific primers and direct sequencing with the ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA)19 (link). Genotyping of CYSLTR1 -634C > T gene polymorphism (rs321029) was performed as previously described, using a primer extension method with a SNaPshot ddNTP primer extension kit (Applied Biosystems)20 (link).
High-resolution typing for HLA-DBP1*0301 was performed as previously described, using the amplified gene product of locus specific primers and direct sequencing with the ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA)19 (link). Genotyping of CYSLTR1 -634C > T gene polymorphism (rs321029) was performed as previously described, using a primer extension method with a SNaPshot ddNTP primer extension kit (Applied Biosystems)20 (link).
3
SNP Identification in SLE Patients
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Fifty SLE patients and 50 NC Korean volunteers were used for SNP identification. Genomic DNA was extracted from whole blood using the QuickGene DNA whole blood kit S (Fujifilm Life Science, Tokyo, Japan). The NR1H3 gene located between the promoter region and intron 2 region was amplified by polymerase chain reaction (PCR) with Amfisure PCR Master Mix (GenDEPOT, Barker, TX, USA). The NR1H2 gene located in the promoter region and between the exon 10 region and the 3’ untranslated region were amplified by PCR. We identified possible polymorphisms in the NR1H3 and NR1H2 genes that were screened by direct sequencing (Bionics, Seoul, Korea). A minor allele frequency ≥5% was considered to indicate a SNP. Additionally, SNP genotyping was performed using the SNaPSHOT ddNTP primer extension kit (Applied Biosystems, Foster City, CA, USA) for SLE patients (n = 250) and NC (n = 167) and replication samples (160 SLE patients and 143 NC).
4
Screening for ATG5 and ATG7 SNPs in Korean samples
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Genomic DNA was prepared from peripheral blood samples using Puregene Blood Core Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. We screened for SNPs located on promoter and 5’-untraslated region (UTR) of ATG5 and ATG7 from 40 healthy Korean volunteers by direct sequencing. Three SNPs of ATG5 including –769T>C, –335G>A in promoter region and 8830C>T in 5’UTR, were selected. Two SNPs of ATG7, including –100 A>G in promoter and 25108G>C in 5’UTR, were selected. ATG5 and ATG7 are located on chromosome 6 and 3, respectively. The information of ATG5 and ATG7 polymorphisms are shown in Table 1 . Minor allele frequency of all selected SNPs was over than 5%. Target SNPs were genotypes from all genomic DNA samples of study subjects using a primer extension method and the SNaPshot ddNTP primer extension kit (Applied Biosystems, Foster City, CA, USA) with primers shown in Table 2 .
5
Genotyping ORMDL3 Variant Analysis
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Genomic DNA was isolated from the peripheral blood using the Puregene DNA purification kit (Gentra, Minneapolis, MN, USA), according to the manufacturer’s instructions. Genotyping of ORMDL3 SNP (rs7216389) was performed as previously described, according to the primer extension method using a SNaP shot ddNTP primer extension kit (Applied Biosystems) [27 (link)].
6
Genotyping Polymorphic Sites via SNaPshot
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Single-base extension reaction was used to genotype polymorphic sites. SNaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA, USA) was used for primer extension reactions according to the manufacturer's recommendations. The primer extension reactions were cleaned by the addition of 1 U of shrimp alkaline phosphatase and incubation at 37℃ for 1 hour, followed by 15 minutes at 72℃ for enzyme inactivation. DNA samples, containing extension products, and GeneScan 120 Liz size standard solution were added to Hi-Di formamide (Applied Biosystems), as recommended by the manufacturer. The mixture was incubated at 95℃ for 5 minutes, cooled on ice for 5 minutes, and electrophoresed using the ABI Prism 3100 Genetic Analyzer. Data were analyzed using ABI Prism GeneScan 3.5.1 and Genotyper 3.6 software (Applied Biosystems).
7
Genotyping of PPARγ Gene SNPs
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130 SNPs in PPARγ were selected based on the reference SNP of the bovine PPARγ gene (GenBank No. NC_007320.5) in dbSNP. Genotypes of 10 SNPs were subjected to a preliminary analysis. Primers for the amplification and extension were designed for the single-base extension (SBE) (Vreeland et al., 2002 (link)) by using forward, reverse, and extension primer sequences (Supplementary Table 1 ). Reactions of the primer extension were performed using the SNaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA, USA). To purify reactions of the primer extension, mixtures containing exonuclease I and shrimp alkaline phosphatase were added to the reaction mixture. Samples were cultured at 37°C for 1 h and then inactivated at 72°C for 15 min. The polymerase chain reaction product was mixed with Genescan 120 LIZ standard and HiDi formamide (Applied Biosystems, USA), followed by denaturation at 95°C for 5 min. Electrophoresis was performed using the ABI PRISM 3130XL Genetic Analyzer, and then electrophoresis products were assayed using GeneMapper v.4.0 (Applied Biosystems, USA).
8
SNP Genotyping via SBE-PCR
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The PCR products were purified by using the FastAP Thermosensitive Alkaline Phosphatase and Exonuclease I (Fermentas -Thermo Fisher Scientific, Waltham, MA, USA) to remove unincorporated ddNTPs, incubated at 37 °C for 45 min, followed by a denaturation step at 85 °C for 15 min typed with the SnaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA, USA). The extension primer used in the typing procedure was SBE1: 5′-CCAgTgCTTTAgATg-gTg-3′ (positions 75 to 82); SBE2 5′-TGAgTATTA-ACCCCAgCTCTAg-3′, (positions 108 to 129); and SBE3 5′-ATTAgTTATTTTAAgATACAgTCTC-3′ (positions 134 to 138) with regard to LPL gene sequence no. GQ150554.1. For electrophoresis, 0.5 µl of purified multiplex SBE reaction was mixed with 0.5 µl of GeneScan-120 LIZ size standard (Applied Biosystems, Foster City, CA, USA) and 10 µl of Hi-Di TM Formamide (Applied Biosystems, Foster City, CA, USA) and denatured at 95 °C for 5 min. The samples were subsequently electrophoresed on an ABI PRISM 3130-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA), after which the data were analyzed and evaluated using GeneMapper ver. 3.5 software (Applied Biosystems, Foster City, CA, USA).
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