Genotyping

HiDi 2x PCR Master Mix

is a ready to use reaction mix that contains all components necessary for PCR based genotyping. Only target specific primers and template need to be added.

 

The included HiDi DNA polymerase is an engineered DNA polymerase variant, specially developed for all approaches in which high single nucleotide discrimination is required, for instance in allele-specific amplifications (ASA) by PCR and primer extensions or methylation-specific PCRs (MSP). HiDi DNA polymerase efficiently amplifies from primers that are matched at the 3'-end and discriminates primers that are mismatched. The kit comes together with an optimized buffer system.

 

HiDi 2x PCR Master Mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes. Carry-over contamination in PCR can be controlled through an enzymatic DNA digestion by adding Uracil-DNA glycosylase (UDG). It can also be used for real-time cycling, when adding a suitable real-time dye or a fluorescent probe. However, it can not be used for hydrolysis-based probes (e.g. TaqMan). 

 

 

Article Product Size Price
┬á#9101 S ┬áHiDi 2x PCR Master Mix 100 reactions ├á 25 ┬Ál, 1 x 1.25 ml ┬áÔéČ 80,-
┬á#9101 M ┬áHiDi 2x PCR Master Mix 500 reactions ├á 25 ┬Ál, 5 x 1.25 ml ┬áÔéČ 360,-

Bulk offer: Should you require larger amounts (> 10 ml), significant discounts are available. Please contact us to receive an individual quotation.

download-pdf Product manual (PDF) Order

 

 

Applications

Applications

  • SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR
  • Genotyping & genomic profiling
  • HLA genotyping
  • Methylation specific PCR (MSP)
  • Multiplex PCR
  • Real-time PCR with a suitable real-time dye
  • End-point PCR

Reaction Setup

Reaction Setup

Component Volume Final concentration
HiDi 2x PCR Master Mix 12.5 ┬Ál ┬á1x
Primer forward (10 ┬ÁM)* 0.5 ┬Ál ┬á0.2 ┬ÁM (0.05-1 ┬ÁM)
Primer reverse (10 ┬ÁM)* 0.5 ┬Ál ┬á0.2 ┬ÁM (0.05-1 ┬ÁM)
Template/Sample extract x ┬Ál  
Nuclease-free water┬á up to 25 ┬Ál total reaction volume  

Keep all components on ice.
Spin down and mix all solutions carefully before use.
* Primers should ideally habe a GC-content of 40-60% 

Typical PCR protocol

Typical PCR protocol

Component Volume Final concentration
Initial denaturation 95┬░C 3 min
Denaturation 95┬░C 10 sec
Annealing* 54-72°C 15 sec     (30-45 cycles)
Extension 72 °C 30 sec / 250 bp
Hold <10┬░C hold

*Typically, the annealing temperature is about 3-5┬░C below the calculated melting temperature of the primers used.

All times may vary depending on the PCR machine.

 

 

Example Results

Example Results

There's no accounting for taste.Abbildung-HiDi1

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/┬Ál of HeLa gDNA┬áin 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. A specific product is visualized by ethidium bromide staining at the right amplicon length of 109 bp.

 

Detailed Product Description

HiDi DNA polymerase is a highly selective DNA polymerase variant. It has been selected for assays in which high discrimination is required, for instance in allele-specific PCRs or methylation-specific PCRs.

 

Whereas many DNA polymerases tolerate mismatched primer-template complexes, HiDi DNA polymerase efficiently discriminates mismatches at  the 3'-primer end and produces specific amplicons in case of perfectly matched primer pairs. The kit comes together with an optimized buffer system.

 

This makes HiDi DNA polymerase very usefull for SNP detections, HLA genotyping or the analysis of single CpG methylation sites.

 

We recommend designing primers with a short amplicon length (about 60-200 bp) for best results, but also longer amplicon lengths are possible. 

HiDi 2x PCR Master Mix can also be used for real-time cycling, when adding a suitable real-time dye or a fluorescent probe. Carry-over contamination in PCR can be controlled through an enzymatic DNA digestion by adding Uracil-DNA glycosylase (UDG). A balanced mix of dUTP and dTTP is already included.

 

Composition / Content of the Product

HiDi 2x PCR Master Mix contains all components necessary for PCR based genotyping including an engineered DNA polymerase, an optimized reaction buffer, and ultrapure dNTPs. An aptamer-based hot-start formulation of the included DNA polymerase prevents false amplification. Temperatures above 50┬░-55┬░C cause the aptamerÔÇÖs secondary structure to melt and will set-free the polymerase.

 

Only target specific primers and template need to be added. Please note that the mix contains a balanced mix of dUTP and dTTP guaranteeing high activity and giving the possibility for UDG based carry-over contamination prevention.

 

Storage

This product is shipped in cool packs. Please store the product upon arrival at - 20 ┬░C. Minimize the number of freeze-thaw cycles by storing in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4 ┬░C.

 

Quality Control

PCR activity: HiDi 2x PCR Master Mix was tested for successful ASA performance detecting a genomic SNP (rs72921001) in HeLa genomic DNA. PCR products were subsequently analysed on a 2.5% agarose gel. A specific product is visualized by ethidium bromide staining at the right amplicon length of 109 bp for the matched primer. In the case of the mismatch primer no product formation is visible after 50 cycles.

DNA polymerase activity: HiDi DNA polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and a DNA primer.

Enzyme-concentration has been determined by protein-specific staining. Please inquire more information at info@mypols.de for the lot-specific concentration.

No contamination has been detected in standard test reactions.

 

 

HiDi

 

img-faqsFAQ`s

Can I use HiDi DNA polymerase with bisulfite-treated DNA samples?

Yes, you can. HiDi DNA polymerase is for example performing very well in methylation specific PCR (MSP). Since a single mismatch is sufficient for specific MSP results, HiDi DNA polymerase even enables reliable MSP analysis of single CpG sites. 

Can I use HiDi 2x PCR Master Mix in probe-based assays?

Yes you can use it in probe assays, which don't require a nuclease activity (e.g. HybProbe or SimpleProbe. Please be aware that HiDi DNA polymerase doesn't have a 5'-3'-nuclease activity, so you can't use HiDi 2x PCR Master Mix in hydrolysis-based probe assays (e.g. TaqMan).

What is the error-rate of HiDi DNA polymerase?

HiDi's error-rate is comparable to the error-rate of wildtype Taq DNA polymerase.

Where does the name HiDi come from?

HiDi stands for High Discrimination. The pronunciation is an allusion to the famous children's TV series 'Heidi, Girl of the Alps, as myPOLS Biotec is located at the northern foot of the Alps, close to 'Heidi-land'

I see a low-molecular band on my agarose gel - is this a contamination of the HiDi 2x PCR Master Mix?

All our products are regularly checked for contamination of various kinds. However it may be possible that the aptamer, which is used for hotstart formulation may be detected as a low molecular band between 25-60 bp length.
Please contact us, should you still have reasonable doubts about the quality of your product. We are always keen to help.

Where can I read more about HiDi DNA polymerase?

HiDi DNA polymerase is based on scientific results, which are published in the open access and peer-reviewed Journal PLOS ONE: 

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification for high discrimination.


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