HiDi® 2x PCR Master Mix
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HiDi® stands for High Discrimination of mismatches at the 3’-terminus of primers in PCR. This myPOLS Biotec enzyme family is optimized for this feature and therefor, is the first choice for applications that rely on this property such as allele-specific PCR (asPCR) that is also termed allele-specific amplification (ASA).
HiDi® 2x PCR Master Mix - ready to use mix simplifies your PCR setup. Only target-specific primers and template need to be added as the mix contains all components for a successful and reliable PCR. This ensures reproducible results, significantly reduces set-up times and the risk of pipetting errors.
This PCR mix is also available with a full-length Taq DNA polymerase with a nuclease domain, featuring 100% compatibility with hydrolysis probes (TaqMan® probes etc.).
Benchmarking with products of competitors conducted by us and others show that the HiDi® DNA polymerase family is the first choice for highly selective PCRs, such as genotyping by allele-specific PCR, HLA genotyping, analysis of single CpG methylation sites or the detection of mutations in a high background of wild-type sequences. By using HiDi® DNA polymerase, less than 10 copies of a mutation can be detected in a background of >10.000 wild-type copies straight away without any other tedious assay optimization.
It has also been shown that HiDi® DNA polymerase family is highly suitable for quality control in CRISPR-Cas or TALEN-based applications.
Several independently conducted studies show that HiDi® DNA polymerase is ideally suited for use in asPCR in numerous research areas ranging from mutation detection to genome editing. (read more)
For research use and further manufacturing.
In case you are aiming to use our RUO products as components or for your development of e.g. an IVD medical device, please contact us.
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What should I consider?
- This mix contains an engineered DNA polymerase in an optimized reaction buffer and ultrapure dNTPs. HiDi® DNA polymerase is hotstart-formulated with an aptamer. Temperatures above 50-55°C cause the aptamer’s secondary structure to melt and will set-free the polymerase.
- HiDi® DNA polymerase can also be used for real-time cycling, when adding a suitable real-time dye, for example GreenDye (#2000). For hydrolysis probe based assays, please refer to our HiDi Taq DNA polymerase or our HiDi Taq 2x PCR master mix.
How do I have to store the mix?
- Keep at -20°C
- Minimize the number of freeze-thaw cycles by storing the product in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C. -
Applications:
- allele-specific PCR (asPCR), allele-specific amplification (ASA)
- HLA genotyping
- analysis of single CpG methylation sites by PCR (methylation specific PCR, MSP)
- mutation detection by PCR even in a high background of wild-type sequences
- genotyping e.g., in CRISPR/Cas and TALEN approaches
Download: HiDi® DNA Polymerase - Application note
Publications:
Lifestyle-specific S-nitrosylation of protein cysteine thiols regulates Escherichia coli biofilm formation and resistance to oxidative stress.
"... After 3–4 cycles, single clones were isolated and screened by PCR using a high discrimination HiDi polymerase (myPols) and primers with the targeted original or mutated nucleotide at the 3′ end (see Table S5). ..."
Barraud N, Létoffé S, Beloin C, Vinh J, Chiappetta G, Ghigo JM.NPJ Biofilms Microbiomes. 2021 Apr 13;7(1):34.
Link to publication: https://doi.org/10.1038/s41522-021-00203-w
Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea.
"…At position 6.97 kb, SNP-specific amplification was performed with HiDi DNA polymerase (myPOLS Biotec, Konstanz, Germany)…"
Miotto O, Sekihara M, Tachibana SI, Yamauchi M, Pearson RD, Amato R, Gonçalves S, Mehra S, Noviyanti R, Marfurt J, Auburn S, Price RN, Mueller I, Ikeda M, Mori T, Hirai M, Tavul L, Hetzel MW, Laman M, Barry AE, Ringwald P, Ohashi J, Hombhanje F, Kwiatkowski DP, Mita T.
PLoS Pathog. 2020 Dec 15;16(12):e1009133.
Link to publication: https://doi.org/10.1371/journal.ppat.1009133
Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.
"…To identify the HDR-targeted clones by SNMD-PCR, the genomic regions surrounding the target loci were amplified using HiDi DNA polymerase (myPOLS Biotec GmbH, Germany) and the corresponding single-nucleotide marker-specific or allele-specific primer pair…"
Yokouchi Y, Suzuki S, Ohtsuki N, Yamamoto K, Noguchi S, Soejima Y, Goto M, Ishioka K, Nakamura I, Suzuki S, Takenoshita S, Era T.
Sci Rep. 2020 Aug 18;10(1):13927.
Link to publication: https://doi.org/10.1038/s41598-020-70401-7
Development of sake yeast haploid set with diverse brewing properties using sake yeast strain Hiroshima no. 6 exhibiting sexual reproduction.
"...MASA typing was performed using high single-nucleotide discrimination (HiDi) Taq DNA polymerase (myPOLS Biotec, Konstanz, Germany)…"
Yamasaki R, Goshima T, Oba K, Kanai M, Ohdoi R, Hirata D, Akao T.
J Biosci Bioeng. 2020 Jun;129(6):706-714.
Link to publication: https://doi.org/10.1016/j.jbiosc.2020.01.005
Influence of EGFR-activating mutations on sensitivity to tyrosine kinase inhibitors in a KRAS mutant non-small cell lung cancer cell line.
"…To detect knock-in mutations, HiDi DNA Polymerase (myPOLS, Konstanz, Germany, US) was used for PCR…"
Tsukumo Y, Naito M, Suzuki T.
PLoS One. 2020 Mar 4;15(3):e0229712.
Link to publication: https://doi.org/10.1371/journal.pone.0229712
Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems.
"…Indeed, with HiDi DNA polymerase, SNP discrimination is outstanding…"
Yang Z, Le JT, Hutter D, Bradley KM, Overton BR, McLendon C, Benner SA.
Biol Methods Protoc. 2020 Feb 10;5(1):bpaa004.
Link to publication: https://doi.org/10.1093/biomethods/bpaa004
Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation.
"…For some chimeric fetuses, allele-specific PCR using Hi-Di DNA polymerase (myPOLS Biotec, Konstanz, Germany) was performed using allele-specific primers, which allowed to amplify the KDR-KO allele (host embryo derived) efficiently…"
Matsunari H, Watanabe M, Hasegawa K, Uchikura A, Nakano K, Umeyama K, Masaki H, Hamanaka S, Yamaguchi T, Nagaya M, Nishinakamura R, Nakauchi H, Nagashima H.
Stem Cell Reports. 2020 Jan 14;14(1):21-33
Link to publication: https://doi.org/10.1016/j.stemcr.2019.11.008
A New Protocol for the Detection of Sterigmatocystin-producing Aspergillus Section Versicolores Using a High Discrimination Polymerase.
"…Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP-based PCR amplification)…"
Kubosaki A, Kobayashi N, Watanabe M, Yoshinari T, Takatori K, Kikuchi Y, Hara-Kudo Y, Terajima J, Sugita-Konishi Y.
Biocontrol Sci. 2020 Jan;25(2):113-118
Link to publication: https://doi.org/10.4265/bio.25.113
CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.
"…Single nucleotide substitutions with dsDNA via HDR mediated by CRISPR-Cas3 and -Cas9 were confirmed by HiDi PCR and sequencing in edited 293T cells…"
Morisaka H, Yoshimi K, Okuzaki Y, Gee P, Kunihiro Y, Sonpho E, Xu H, Sasakawa N, Naito Y, Nakada S, Yamamoto T, Sano S, Hotta A, Takeda J, Mashimo T.
Nat Commun. 2019 Dec 6;10(1):5302
Link to publication: https://doi.org/10.1038/s41467-019-13226-x
END-phenomenon negative bovine viral diarrhea virus that induces the host's innate immune response supports propagation of BVDVs with different immunological properties.
"…Because vBVD12− and vBVD12−/P8L differ only by a one-base mutation in the region encoding Npro (at nucleotide 410), it is possible to discriminate the sequences of the respective viruses using HiDi DNA polymerase…"
Shiokawa M, Omatsu T, Katayama Y, Nishine K, Fujimoto Y, Uchiyama S, Kameyama KI, Nagai M, Mizutani T, Sakoda Y, Fukusho A, Aoki H.
Virology. 2019 Dec;538:97-110
Link to publication: https://doi.org/10.1016/j.virol.2019.09.016
Loss of ALBINO3b Insertase Results in Truncated Light-Harvesting Antenna in Diatoms.
"…Both alleles were amplified separately using HiDi polymerase (myPols) according to the manufacturer’s instructions…"
Nymark M, Volpe C, Hafskjold MCG, Kirst H, Serif M, Vadstein O, Bones AM, Melis A, Winge P.
Plant Physiol. 2019 Nov;181(3):1257-1276
Link to publication: https://doi.org/10.1104/pp.19.00868
Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum.
"…PCR was performed using HiDi polymerase according to the manufacturer’s instructions for 30 cycles (MyPols, Germany), with either the primer pairs Lhcx1_wild-type-fw/Lhcx1_wild-type-rev and Lhcx1_all-fw/Lhcx1-all_rev or with Lhcx1_comp-fw/Lhcx1_wild-type-rev and Lhcx1_all-fw/Lhcx1-all_rev…"
Buck JM, Sherman J, Bártulos CR, Serif M, Halder M, Henkel J, Falciatore A, Lavaud J, Gorbunov MY, Kroth PG, Falkowski PG, Lepetit B.
Nat Commun. 2019 Sep 13;10(1):4167
Link to publication: https://doi.org/10.1038/s41467-019-12043-6
Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.
"…Notably, under the stringent conditions (where Mg2+ concentration was lowered), HiDi DNA polymerase tended to show superior performance relative to rTaq DNA polymerase in terms of the inability to amplify samples harbouring indels…. Furthermore, HiDi DNA polymerase was also superior in terms of the ability to detect the WT samples among samples enriched with mosaic mutations…"
Sakurai T, Kamiyoshi A, Takei N, Watanabe S, Sato M, Shindo T.
Sci Rep. 2019 Jul 9;9(1):9923
Link to publication: https://doi.org/10.1038/s41598-019-46357-8
A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum
S Madhuri, CR Bártulos, M Serif, B Lepetit, PG Kroth
Algal Research, 2019 May, 101469
Link to publication: https://doi.org/10.1016/j.algal.2019.101469
GPR31-dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial metabolites.
"…To distinguish the difference in the expression between Gpr31b and other Gpr31 isoforms, PCR was performed with HiDi polymerase (myPOLS Biotec), which discriminates primers with a mismatch at the 3′-end…"
Morita N, Umemoto E, Fujita S, Hayashi A, Kikuta J, Kimura I, Haneda T, Imai T, Inoue A, Mimuro H, Maeda Y, Kayama H, Okumura R, Aoki J, Okada N, Kida T, Ishii M, Nabeshima R, Takeda K.
Nature. 2019 Feb;566(7742):110-114
Link to publication: https://doi.org/10.1038/s41586-019-0884-1
Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair.
"…We further confirmed gene editing by PCR using high single-nucleotide discrimination (HiDi) DNA polymerase that efficiently discriminates primers with a mismatch at the 3′ end … The edited alleles, but not non-edited alleles, can be amplified by HiDi PCR using the edited allele-specific PCR primer…. "
Nakajima K, Zhou Y, Tomita A, Hirade Y, Gurumurthy CB, Nakada S.
Genome Res. 2018 Feb;28(2):223-230
Link to publication: https://doi.org/10.1101/gr.226027.117
One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.
"…The highly selective HiDi DNA polymerase (myPOLS, Germany) was used for allele-specific PCR, together with primers ending with single nucleotide polymorphisms..."
Serif M, Dubois G, Finoux AL, Teste MA, Jallet D, Daboussi F.
Nat Commun. 2018 Sep 25;9(1):3924
Link to publication: https://doi.org/10.1038/s41467-018-06378-9
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HiDi® 2x PCR Master Mix is tested for successful allele-specific PCR detecting a genomic SNP (rs72921001) in HeLa genomic DNA. In case of the mismatch primer no specific product formation was observed after 50 cycles.
The activity of HiDi® DNA polymerase is monitored and adjusted to a specific DNA polymerase activity using a synthetic DNA template and DNA primer. Enzyme concentrations are determined by protein-specific staining. Please inquire more information using our contact form for lot-specific concentrations. No contamination is detected in standard test reactions
Example Primer Design
Matching vs. mismatching nucleotide is placed at the 3'-end of the primer for best discrimination results.
Example Results - There´s no accounting for taste
Cilantro: some people love it in their food, some hate it. Here we are detecting a genomic SNP (rs72921001) in HeLa genomic DNA. This SNP is reported to be close to a number of genes coding for olfactory receptors. (Reference: Eriksson N. et al. (2012), "A genetic variant near olfactory receptor genes influences cilantro preference.")
Considering, that only the C-allele specific primer is extended and yielding in a specific amplicon, we can conclude a genetic predisposition in disliking cilantro, as this SNP is significantly associated with detecting a soapy taste to cilantro.
Allele-specific PCRs were performed from 1 ng/µl of HeLa gDNA in the presence of a realtime dye, indicating the amplification of the C-allele specific primer only. The A-allele specific primer is discriminated, thus not amplified up to 50 cycles.
PCR products were subsequently analysed on a 2.5% agarose gel. Specific product is visualized by ethidium bromide staining at the amplicon length of 109 bp.
