Hotstar polymerase

Manufactured by Qiagen

Sourced in Germany

HotStar polymerase is a heat-activated DNA polymerase used in PCR (Polymerase Chain Reaction) applications. It exhibits robust performance and high specificity in amplifying DNA targets.

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Lab products found in correlation

Dneasy kit, by Qiagen (1 mentions) Dntp mix, by Roche (1 mentions) Pcr buffer, by Qiagen (1 mentions) Mgcl2, by Qiagen (1 mentions) Superscript 2, by Thermo Fisher Scientific (1 mentions) Ez dna methylation gold kit, by Zymo Research (1 mentions) Epidesigner, by Agena (1 mentions) Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)

8 protocols using hotstar polymerase

Allele-Specific Genotyping of SNPs

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Genotyped SNPs were identified from Rogaeva et al. (2007) (link) and are listed in Figure 1.
Fibroblast cultures from each patient were lysed, and genomic DNA was isolated using the DNeasy kit (QIAGEN). Allele-specific primers for the SNPs analyzed were designed using the Web-based allele-specific primer-designing tool (http://bioinfo.biotec.or.th/WASP). PCR were run using Hot-Star Polymerase (QIAGEN) and analyzed by agarose gel electrophoresis. A list of all allele-specific primers used in this study is provided in the Supplemental Experimental Procedures.

Young J.E., Boulanger-Weill J., Williams D.A., Woodruff G., Buen F., Revilla A.C., Herrera C., Israel M.A., Yuan S.H., Edland S.D, & Goldstein L.S. (2015). Elucidating Molecular Phenotypes Caused by the SORL1 Alzheimer’s Disease Genetic Risk Factor Using Human Induced Pluripotent Stem Cells. Cell stem cell, 16(4), 373-385.

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HEV Genome Amplification Protocol

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HEV primer design was based on gt3 genomes with accession numbers FJ705359 and FJ956757.1 [32 (link)]. cDNA of HEV0069 infected samples were used to generate seven overlapping amplicons of ~2000 bp length each (Figure S2). Subsequently, two or three 500 bp long amplicons, overlapping ~50–100 bp, per primary product were generated using nested primers to cover the full HEV genome (Table S1A,B). Each 50µL reaction contained 10 µL HEV cDNA, 0.4 µM primers, 1× PCR buffer (Qiagen), 1 mM MgCl₂ (Qiagen), 0.2 mM dNTP mix (Roche), and 2.5 U HotStar polymerase (Qiagen). For PCR, the conditions were 15 min 95 °C, 40 cycles of 1 min 95 °C, 1 min 50 °C, 3 min 72 °C, and 10 min 72 °C. 2 μL first PCR product served as template for the nested PCR. The PCR conditions were identical to first round PCR, with 40 cycles of 30 sec 95 °C, 30 sec 50 °C, and 1 min 72 °C. Amplicon 15 was amplified using an altered annealing temperature of 45 °C instead of 50 °C.
In order to amplify cDNA from serum and feces-derived HEV0122, additional primer pairs were adapted from Munoz-Chimeno et al. [33 (link)] as shown in Table S1C,D. PCR conditions were identical as described above, with minor modifications: 5 μL cDNA was used as input for the first round PCR, and 2.5 μL first PCR product served as template for nested PCR. The annealing temperatures during the PCR protocols were adjusted to match each primer set.

Sari G., van de Garde M.D., van Schoonhoven A., Voermans J.J., van der Eijk A.A., de Man R.A., Boonstra A., Vanwolleghem T, & Pas S.D. (2019). Hepatitis E Virus Shows More Genomic Alterations in Cell Culture than In Vivo. Pathogens, 8(4), 255.

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Avian Species Identification by PCR

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Sequence-specific PCR was conducted with HotStar polymerase (Qiagen) and random hexamer-primed cDNA (Superscript II, Invitrogen). Amplified products were subjected to direct dideoxy sequencing (Genewiz, South Plainfield, NJ, USA). The bird species from which samples were obtained was identified by the nested PCR method of Cheung et al. that targets the mitochondrial cytochrome oxidase I (COI) gene [9] (link).

Honkavuori K.S., Briese T., Krauss S., Sanchez M.D., Jain K., Hutchison S.K., Webster R.G, & Lipkin W.I. (2014). Novel Coronavirus and Astrovirus in Delaware Bay Shorebirds. PLoS ONE, 9(4), e93395.

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Pyrosequencing-based Methylation Analysis of CpG Sites

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The methylation status of the selected CpGs was validated in 87 subjects (Figure 1, Supplemental Table S1) by pyrosequencing bisulfite-treated DNA. Genomic DNA was extracted as described above and 1µg of DNA was bisulfite using the EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA, USA). Bisulfite-treated DNA (40 ng) was amplified with 0.2 µM of forward and biotinylated-reverse primers (Supplemental Table S2). Reactions were conducted in 1X Hot Star Buffer, 0.2 mM dNTPs, and 0.8 U of Hot Star Polymerase (Qiagen, Hilden, Germany) in a total volume of 20 µL. PCR cycling steps were: 15 min at 95 °C followed by four cycles of 20 s at 95 °C, 30 s at 65 °C and 60 s at 72 °C; later four cycles of 20 s at 95 °C, 30 s at 58 °C, 60 s at 72 °C; and 38 cycles of 20 s sat 95 °C, 30 s at the annealing temperature of each pair of primers (Supplemental Table S2), 60 s at 72 °C; to finish 3 min at 72 °C. The PCR product was rendered single-stranded and pyrosequenced in a PyroMark Q96 IS (Qiagen, Hilden, Germany) at Instituto de Investigación Biomédica de Málaga (IBIMA, Málaga, Spain) with 4 pmol of the sequencing primer. Raw data were analyzed using the Pyromark CpGs software (Qiagen, Hilden, Germany), and the percentage of methylation for each analyzed CpGs was obtained.

Mas-Parés B., Xargay-Torrent S., Gómez-Vilarrubla A., Carreras-Badosa G., Prats-Puig A., De Zegher F., Ibáñez L., Bassols J, & López-Bermejo A. (2023). Gestational Weight Gain Relates to DNA Methylation in Umbilical Cord, Which, In Turn, Associates with Offspring Obesity-Related Parameters. Nutrients, 15(14), 3175.

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ABCB1 Genotyping: ARMS-PCR and KASP

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The variant rs2032582 of ABCB1 gene was genotyped using the amplification-refractory mutation system polymerase chain reaction (ARMS PCR). A forward primer was designed for each allele specifically in order to pinpoint the exact genotype. The protocol was adapted from Kuzawski and coworkers.30 (link) For each patient’s sample, 3 separate PCR mixes were prepared, each containing different allele specific primer. In the final reaction volume of 15 microL, 1x buffer were added, 3 mM of MgCl₂, 0.67 mM of dNTP, 0.3 microM of the forward (allele specific) and reverse primer, 1U of Taq polymerase (Hot Star polymerase, Qiagen, Hilden, Germany) and 60ng of DNA. The PCR program started with a 95 °C initial denaturation which lasted for 15′, followed by 10 cycles of 95 °C / 60 °C / 72 °C lasting 30″, 30″ and 40″ respectively, followed by 30 cycles of 95 °C / 56 °C / 72 °C, lasting 30″, 30″ and 40″ respectively and the final step was an extension at 72 °C which endured for 5′.
Variants rs1045642 and rs1128503 of ABCB1 were genotyped using the Kompetitive Allele Specific PCR genotyping system (KASP) (LGC, Teddington, Middlesex, UK), according to manufacturer’s instructions.

Gasic V., Zukic B., Stankovic B., Janic D., Dokmanovic L., Lazic J., Krstovski N., Dolzan V., Jazbec J., Pavlovic S, & Kotur N P.h.D. (2018). Pharmacogenomic Markers of Glucocorticoid Response in The Initial Phase of Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia. Radiology and Oncology, 52(3), 296-306.

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Amplicon Bisulfite Sequencing for CXCL10 Methylation

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Amplicon bisulfite sequencing was used to measure methylation at 7 CpG sites in the promoter of the CXCL10 gene. Primers were designed using the EpiDesigner tool from Agena Biosciences (EpiDesigner.com">www.EpiDesigner.com). Genomic DNA was bisulfite converted, and amplicons were amplified using Qiagen HotStar polymerase. For each patient, amplicons were pooled at equimolar concentration before preparing libraries using the KAPA Hyper Prep library kit. Final libraries were quantified using the KAPA Library prep quantification kit, pooled at equimolar concentration, and sequenced on a MiSeq Nano V2 flow cell with PE-150 cycles. The data generated was trimmed of adapter sequences, mapped to the human genome (hg38) using Bismark, and methylation data was extracted using MethylDackel. The resulting files contain methylation data as beta values for each CpG site/sample. These were imported in R for analysis.

Zakharia Y., Singer E.A., Acharyya S., Garje R., Joshi M., Peace D., Baladandayuthapani V., Majumdar A., Li X., Lalancette C., Kryczek I., Zou W, & Alva A. (2024). Durvalumab and guadecitabine in advanced clear cell renal cell carcinoma: results from the phase Ib/II study BTCRC-GU16-043. Nature Communications, 15, 972.

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Molecular Genotyping of Neisseria gonorrhoeae

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PCR for both targets was performed in 50 µL reaction volumes using the Biometra T3000 Thermal Cycler (Labrepco Inc., US). Each reaction per target (porB and tbpB) contained 50 pmol of the NG-MAST forward and reverse primer for the respective target (Table 1), 2.5 U HotStar polymerase (Qiagen), 1× Qiagen PCR buffer, 0.2 mmol/L dNTP, 5 µL DNA lysate and MQ to a volume of 50 µL. The PCR protocol of Martin et al. was used to amplify the targets but cycles were increased to 30 [7 (link)].
The amplicons were precipitated with 50 µL 20% polyethylene glycol 8000 and 2.5 mol/L sodium chloride at 37 °C for 15 min. Precipitated amplicons were centrifuged at 15,000 × g for 15 min and washed twice with ice-cold 80% ethanol. The pellet was allowed to dry and resuspended in 25 µL MQ.
The porB and tbpB fragments were sequenced with their respective forward and reverse primer using the BigDye Terminator v1.1 Cycle Sequencing kit (Thermo Fisher Scientific, Waltham, Massachusetts, US). The sequence protocol has an initial denaturation step of 1 min at 96 °C, followed by 25 cycles of 10 s at 96 °C, 10 s at 55 °C (porB) or 65 °C (tbpB), and 3 min at 60 °C.

van der Veer B.M., Wolffs P.F., Hoebe C.J., Dukers-Muijrers N.H, & van Alphen L.B. (2018). Culture-free genotyping of Neisseria gonorrhoeae revealed distinct strains at different anatomical sites in a quarter of patients, the Netherlands, 2012 to 2016. Eurosurveillance, 23(50), 1800253.

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Multiplex PCR for GSTM1 and GSTT1 Genotyping

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The detection of GSTM1 and GSTT1 homozygous deletions was performed using multiplex polymerase chain reaction (PCR), as previously described with modifications.29 (link) In the final reaction volume of 20 microL, 1x buffer were added, 3.875 mM of MgCl₂, 0.5 mM of dNTP, 0.3 microM of the forward and reverse primer for GSTT1, 0.25 microM of the forward and the reverse primer for GSTM1, 0.25 microM of forward and reverse primer for β globin gene segment (control PCR product), 1U of Taq polymerase (Hot Star polymerase, Qiagen, Hilden, Germany) and 60ng of DNA. After the initial denaturation at 95 C for 15′, followed 35 cycles of 95 °C / 53 °C / 72 °C, lasting 30″, 45″ and 60″ respectively, ending with a final extension step at 72 °C lasting for 7′.

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