Dna isolation kit

Manufactured by Roche

Sourced in Germany, United States

The DNA Isolation Kit is a laboratory instrument designed to extract and purify DNA from various biological samples. It utilizes a series of buffers and columns to selectively capture and wash DNA, allowing for the isolation of high-quality genetic material for further analysis and applications.

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29 protocols using dna isolation kit

Pharmacogenomic Profiling of Japanese Patients

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SNPs at CYP3A4, CYP3A5, OATP1B1, OATP2B1, and P-gp, with minor allele frequencies >0.1 among the Japanese population, were selected based on the HapMap Japanese in Tokyo database. Among these, tag SNPs were further determined using PLINK version 1.9 and Haploview version 4.2 software (Broad Institute, Cambridge, MA, USA). Pairwise linkage disequilibrium statistics, estimated frequencies of haplotype, and haplotype blocks were also calculated using these software packages. Hardy–Weinberg equilibrium was assessed using chi-square test with one degree of freedom. In addition, uridine diphosphate glucuronosyltransferase family 1 member A1 (UGT1A1) rs 4148323 was determined, because this SNP is reportedly associated with ASV/DCV-induced ALT elevation [5 (link)].
Genomic DNA was extracted from whole blood using the MagNA Pure LC and the DNA Isolation Kit (Roche Diagnostics). Tag SNPs were genotyped by real-time detection PCR using the TaqMan SNP Genotyping Assays or rhAmp SNP Genotyping System (IDT, Coralville, IA, USA) and the LightCycler 96 System (Roche Diagnostics).

Kato K., Shimada N., Atsukawa M., Abe H., Itokawa N., Matsumoto Y., Agata R, & Tsubota A. (2019). Single nucleotide polymorphisms associated with elevated alanine aminotransferase in patients receiving asunaprevir plus daclatasvir combination therapy for chronic hepatitis C. PLoS ONE, 14(7), e0219022.

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Genotyping eNOS Polymorphism in Venous Blood

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Venous blood samples were taken from all patients before surgeries and stored at + 4°C in sterile EDTA tubes until the planned analyses were performed. DNA samples were isolated from blood samples using DNA isolation kit (Cat No: 1796828, Roche Diagnostics, GmbH, Mannheim, Germany) in accordance with the procedures. To evaluate the 894 G>T (rs1799983) polymorphism on the eNOS gene, the TaqMan® SNP Genotyping Assay (Cat No: 4351379, NOS3 rs1799983, Thermo Fisher Scientific, USA) kit was used in accordance with the procedures recommended by the company. 15 μL of reaction mixture (Master Mix, Primer-probe Assay, H2O) and 5 μL of DNA were added to the multiwell plate for each sample. Analysis was performed on RT-PCR (LC480, Roche) (Table 1).

Dursun O., Yilmaz A., Fidanci S.B., Tamer L., Vatansever M., Ozer O, & Dinc E. (2022). Investigation of eNOS G894T Gene Polymorphism in Patients with Pseudoexfoliation Syndrome: A Preliminary Study. Beyoglu Eye Journal, 7(3), 188-192.

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Genotyping of rs8099917 SNP in IL-28B Gene

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Genomic DNA was extracted from whole blood using MagNA Pure LC and the DNA Isolation Kit (Roche Diagnostics). The rs8099917 single nucleotide polymorphism (SNP) near the IL-28B gene [19 (link)] was genotyped by RT-PCR using the TaqMan SNP Genotyping Assay and the 7500 Fast RT-PCR System (Applied Biosystems, Foster City, CA, USA). The rs8099917 genotype was classified into 2 categories: TT (major genotype) and non-TT genotype (minor genotype, TG or GG).

Abe H., Tsubota A., Shimada N., Atsukawa M., Kato K., Takaguchi K., Asano T., Chuganji Y., Sakamoto C., Toyoda H., Kumada T., Ide T., Sata M, & Aizawa Y. (2014). Predictors of Response to 24-Week Telaprevir-Based Triple Therapy for Treatment-Naïve Genotype 1b Chronic Hepatitis C Patients. Gastroenterology Research and Practice, 2014, 549709.

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Quantifying HSV-1 Infection in AAV-Transduced HTK Cells

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HTK cells (1 × 10⁵ per well, 24-well plates) were treated with 17 × 10¹⁰ AAV-MEGA or AAV-CTRL vg for 48 h in ABAM-free medium. Then cells were incubated for 1 h at room temperature with serial rHSV-1 dilutions to reach 0.5, 0.05, or 0.005 MOIs and further cultured in ABAM-free medium. After rinsing with Dulbecco’s-phosphate-buffered saline (D-PBS), cultures were continued in ABAM-free medium for 24 h before processing for analysis. (1) A β-Galactosidase Enzyme Assay System (Promega kit #E2000; hydrolysis of its colorless substrate ortho-nitrophenyl-β-galactoside, to o-nitrophenol, a yellow chromophore) was used on total cell protein extract, according to the manufacturer’s instructions, before TECAN-Infinite M1000 measurement of chromophore absorbance. Total protein concentration was estimated by Lowry’s assay (Bio-Rad #500-0112). (2) After cell lysis (50 mM Tris-HCl pH8, 200 mM NaCl, 5 mM EDTA, 0.5% SDS), DNA was isolated using DNA Isolation Kit (Roche Diagnostics #03115 879,001), according to the manufacturer’s instructions. qPCR enabled measurement of UL18 VP5, human β-actin, and carboxypeptidase X1 (Cpxm1) gene expressions.

Chapellier B., Guindolet D., Pereira D., Galetto R., Sahel J.A., Labetoulle M, & Gabison E.E. (2022). Meganuclease targeting HSV-1 protects against herpetic keratitis: Application to corneal transplants. Molecular Therapy. Nucleic Acids, 30, 511-521.

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Peripheral Blood DNA Isolation

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DNA was extracted from the peripheral blood leukocytes using the DNA Isolation Kit (Roche, Indianapolis, USA).

Zhao Y., Zhang Y., Wang X, & Zhang L. (2019). Variant analysis in Chinese families with hereditary hemorrhagic telangiectasia. Molecular Genetics & Genomic Medicine, 7(9), e893.

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DNA Extraction from Tsetse Flies

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Dry whole tsetse flies were transferred to new Eppendorf tubes and crushed using a sterilised plastic pestle. Parasite DNA was extracted from the crushed tsetse flies using Roche DNA isolation kit (Mannheim, Germany) as per the manufacturer’s instructions. The DNA was suspended in 100 µL of PCR water and stored at -20 °C.

Nambala P., Musaya J., Hayashida K., Maganga E., Senga E., Kamoto K., Chisi J, & Sugimoto C. (2018). Comparative evaluation of dry and liquid RIME LAMP in detecting trypanosomes in dead tsetse flies. The Onderstepoort Journal of Veterinary Research, 85(1), 1543.

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Quantitative PCR for Telomere Length

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Quantitative PCR (real-time PCR or qPCR) was used to measure relative telomere lengths (RTL) of ntESCs, as previously described [35 (link)]. Briefly, genomic DNA was extracted from cells using the DNA Isolation Kit (High Pure PCR Template Preparation Kit, 11796828001, Roche, Basel, Switzerland). For each sample, 20 ng of DNA was used in each reaction. PCR reactions were performed on the SYBR Green detection system (KK4603, Kapa Biosystems, Inc., Woburn, MA, USA), using telomeric primers (5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′ and 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′). For each PCR reaction, a standard curve was made by serial dilutions of known amounts of mouse genomic DNA. The telomere signal was normalized to the signal from the single-copy gene (36B4: 5′-ACTGGTCTAGGACCCGAGAAG-3′ and 5′-TCAATGGTGCCTCTGGAGATT-3′) to generate a telomere to single-copy gene ratio (T/S ratio) indicative of relative telomere length of the given sample. Each reaction was performed in triplicate.

Chang W.F., Wu Y.H., Xu J, & Sung L.Y. (2019). Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer. International Journal of Molecular Sciences, 20(5), 1236.

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Genetic Profiling of DRD1 and DRD3

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We drew 5-ml peripheral blood samples from participants at the First Affiliated Hospital of Kunming Medical University and the Human Genetics Center of Yunnan University. Genomic DNA was extracted using the DNA isolation kit (Roche Diagnostics, USA), according to the manufacturer’s instructions. In accordance with the known sequence of human DRD1 and DRD3, 3 primers of DRD1 and 6 primers of DRD3 were designed to amplify the entire coding region. Amplification was performed in 25-μL reaction volume containing 1U AmpliTaq Gold-polymerase, 2 ng genomic DNA, 10 μmol/L of each primer, and PCR assay mix (PE Applied Biosystems, Foster City, CA). All PCR fragments were carried out in 96-well plates on a programmable thermal cycler using the ABI 373 fluorescence PCR system (PE Applied Biosystems, Foster City, CA).

Yang H., Zhong L., Bai S., Dong Y., Wang Y., Li Q., Yang Q., Jiang D., Bi H., Xiao C, & Zheng B. (2017). Association of Dopamine D1 and D3 Receptor Gene Polymorphisms with Essential Hypertension in 3 Ethnic Groups in China. Medical Science Monitor Basic Research, 23, 234-239.

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Quantifying Antibody Gene Copy Number

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Genomic DNA was extracted from 1*10⁶ cells and purified using the DNA isolation kit (Roche). The quality and concentration of DNA were determined measuring absorbance at 260/280 nm. Gene copy number (GCN) of antibody based on the light chain gene was quantified by qRT-PCR. 20 ng genomic DNA of each cell pool was used per reaction. Oligonucleotide sequences are summarized in Table 2. The qRT-PCR run was carried out as following: stage 1, 95°C for 10 min; stage 2, 95°C for 15 sec, 65°C for 40 sec, and 60°C for 35 sec which was repeated 40 times. Each run was conducted in triplicate. Raw data were analyzed as described by Osterlehner et al. [26 (link)].

Ebadat S., Ahmadi S., Ahmadi M., Nematpour F., Barkhordari F., Mahdian R., Davami F, & Mahboudi F. (2017). Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells. PLoS ONE, 12(10), e0185967.

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PD-L1 Methylation Analysis Protocol

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Genomic DNA was isolated from the tumor lines using a DNA isolation kit according to the manufacturer's instructions (Roche Applied Science, Berlin, Germany). PD-L1 methylation status was determined by the methylation-specific (Ms) PCR and pyrosequencing, using the following primers for sequence amplification: for cg15837913: sense, 5′-GGTAGAATATTAGGGATTTTGAGTATTT-3′; anti-sense, 5′-CAACAACAAA CCCATATAACTTTAAT-3′. For cg19724470: sense, 5′-TTGATGTTAGGTTGGAGGT TTG-3; anti-sense, 5′-AAACTCCTCCATTCCTCTTT-3′. The analysis was performed by Beijing Genomics Institute (BGI) in China. The information related to the primers used in this study is displayed in Table S3.

Mu L., Long Y., Yang C., Jin L., Tao H., Ge H., Chang Y.E., Karachi A., Kubilis P.S., De Leon G., Qi J., Sayour E.J., Mitchell D.A., Lin Z, & Huang J. (2018). The IDH1 Mutation-Induced Oncometabolite, 2-Hydroxyglutarate, May Affect DNA Methylation and Expression of PD-L1 in Gliomas. Frontiers in Molecular Neuroscience, 11, 82.

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