Genotyping

HiDi DNA polymerase

is a highly selective DNA polymerase variant, specially evolved for all assays in which high single nucleotide discrimination is required, for instance in allele-specific PCRs (ASA), primer extensions or methylation-specific PCRs (MSP).

HiDi DNA polymerase efficiently discriminates primers, which have a mismatch at the 3'-end.

 

The engineered polymerase is also available as HiDi Taq version which has a 5'-3'-nuclease activity and therefore can be used for hydrolysis probe-based assays.

 

 

Article Product Size Price
┬á#9001 S ┬áHiDi DNA polymerase 250 U, 5U/┬Ál, 50 ┬Ál ┬áÔéČ 70,-
┬á#9001 M ┬áHiDi DNA polymerase 1000 U, 5U/┬Ál, 200 ┬Ál ┬áÔéČ 255,-
┬á#9201 S ┬áHiDi Taq DNA polymerase 250 U, 5U/┬Ál, 50 ┬Ál ┬áÔéČ 70,-
┬á#9201 M ┬áHiDi Taq DNA polymerase 1000 U, 5U/┬Ál, 200 ┬Ál ┬áÔéČ 255,-

Bulk offer: Should you require larger amounts (> 2000 U), significant discounts are available. Please contact us to receive an individual quotation.

download-pdf Product manual Hidi (PDF) Order

download-pdf Product manual Hidi Taq (PDF)

 

 

Applications

Applications

  • SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR
  • Methylation specific PCR (MSP)
  • HLA genotyping
  • Multiplex PCR

HiDi Taq DNA polymerase

  • real-time PCR with fluorescence-based hydrolysis probes
  • real-time multiplex detection PCR

Reaction Setup

Reaction Setup

Component Volume Final concentration
Primer forward (10 ┬ÁM)* ┬á1 ┬Ál ┬á0.2 ┬ÁM (0.05-1 ┬ÁM)
Primer reverse(10 ┬ÁM)* ┬á1 ┬Ál ┬á0.2 ┬ÁM (0.05-1 ┬ÁM)
dNTPs (2 mM) ┬á5 ┬Ál ┬á200 ┬ÁM
10 x HiDi buffer ┬á5 ┬Ál ┬á1x
HiDi DNA polymerase 5 U/┬Ál ┬á0.5 ┬Ál ┬á1.25 - 5 U/reaction
Template/Sample extract ┬áx ┬Ál  
Nuclease-free water┬á ┬áup to 50 ┬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 2 min
Denaturation 95┬░C 15 sec
Annealing* 54-72°C 10 sec     (25-40 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 those and only produces specific amplicons in case of perfectly matched primer pairs. This makes HiDi DNA polymerase very usefull for SNP detections, HLA genotyping or the analysis of single CpG methylation sites.

 

An aptamer-based hot-start formulation of the HiDi DNA polymerase prevents false amplification. Temperatures above 50┬░-55┬░C cause the aptamerÔÇÖs secondary structure to melt and will set-free the polymerase.

 

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

The addition of additional Magnesium (+ 0.5 - 1.5 mM) might be needed in case of longer amplicons >500 bp. HiDi DNA polymerase can also be used for real-time cycling, when adding a suitable real-time dye.

 

HiDi Taq variant has a 5'-3'-nuclease activity and therefore is suitable for hydrolysis probe-based assays.

 

Composition / Content of the Product

HiDi DNA polymerase is supplied as a 5 U/┬Ál solution. It comes together with an┬áoptimized 10x reaction buffer.

 

Storage

This product is shipped in cool packs. Please store the product upon arrival at - 20┬░C.

 

Quality Control

PCR activity: HiDi DNA polymerase 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. HiDi Taq DNA polymerase was succesfully tested for hydrolysis probe based real-time PCR. The product demonstrates linearity of amplification over a specified serial dilution of human genomic DNA.

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 DNA polymerase in real-time PCRs using a real-time dye such as SYBR Green?

Please be aware that HiDi Taq DNA polymerase is not suitable for real-time PCRs using a real-time dye. HiDi DNA polymerase works very well with real-time dyes.

Can I use HiDi DNA polymerase in probe-based assays?

HiDi Taq variant has a 5'-3'-nuclease activity and therefore can be used for hydrolysis probe-based real-time PCRs. 

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/HiDi Taq DNA polymerase?

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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