Genotyping

Direct Oral Swab 2x PCR Master Mix

DNA isolation is not needed anymore -

Direct Oral Swab 2x PCR Master Mix allows direct PCR, as well as PCR based genotyping or pathogen detection directly from saliva or buccal swab samples.

The included DNA polymerase variant (HiDi Taq) is engineered for superior PCR performance directly from saliva or buccal swab samples.

Direct Oral Swab 2x PCR Master Mix contains HiDi Taq DNA polymerase and thus, is also proficient in all assays in which high single nucleotide discrimination is required, for instance in allele-specific amplifications (ASA) by PCR. HiDi Taq 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 and a separate lysis buffer.

The Direct Oral Swab 2x PCR Master Mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes. It can also be used for real-time cycling when using fluorescence-based hydrolysis probes.

 

Article Product Size Price
 #9501 S  Direct Oral Swab 2x PCR Master Mix 100 reactions à 25 µl, 1 x 1.25 ml  € 85,-
 #9501 M  Direct Oral Swab 2x PCR Master Mix 500 reactions à 25 µl, 5 x 1.25 ml  € 380,-

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

 

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Applications

Applications

  • Direct PCR from saliva samples
  • Pathogen detection directly from saliva samples
  • Genotyping & genomic profiling
  • SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR directly from saliva samples
  • HLA genotyping
  • End-point PCR
  • Real-time PCR with with fluorescence-based hydrolysis probes

Reaction Setup

 

Template preparation

Direct Oral Swab PCR Kit has been optimized for various sample collection kits and fresh saliva samples.

 

Sample collection kits

As amounts of stabilizing liquid vary between manufactures it is recommended to dilute the collected sample with the provided lysis buffer to a final concentration of approximately 1-5 % saliva solution e.g.  2 µl sample (20 - 100 % saliva concentration) + 38 µl lysis buffer . As template 2.5 µl of this solution is then directly used for a 25 µl PCR reaction.

 

Fresh saliva 

Fresh saliva should be collected in a reaction tube and centrifuged for 20 sec in a standard benchtop centrifuge at maximum speed. 2 µl of the supernatant should be mixed with 38 µl of the provided lysis buffer (5 % saliva concentration). After brief vortexing the reaction is cleared by centrifugation in a standard benchtop centrifuge for 60 sec at maximum speed. As template 2.5 µl of the supernatant is then directly used for a 25 µl PCR reaction yielding a final saliva concentration of 0.5 %.

 

In the case of DNA collection with buccal swabs the sample should be placed in 200 µl of the provided lysis buffer. As template 2.5 µl of the saliva solution is then directly used for a 25 µl PCR reaction.

 

Please note that the saliva solution of freshly collected samples without a sample collection kit can only be stored for up to 24 h at 4°C before use.

 

Reaction Setup

Component Volume Final concentration
Direct Oral Swab 2x PCR Master Mix  12.5 µl  1x
Primer forward (10 µM)*  0.5 µl  0.2 µM (0.05-1 µM)
Primer reverse(10 µM)*  0.5 µl  0.2 µM (0.05-1 µM)
Template/Sample extract  2.5 µl  0.1- 0.5 %
Nuclease-free water  up to 25 µl total reaction volume  

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

Typical PCR protocol

Typical PCR protocol

Step Temperature Time
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

 

Cilantro: some people love it in their food, some hate it. Here we are detecting a genomic SNP (rs72921001) in human genomic DNA from  saliva samples. 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.")

 

If only the C-allele specific primer is extended yielding 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.

In order to detect the genomic SNP, a 109 bp fragment was amplified from three different human saliva samples, respectively with the C-allele or the A-allele specific primer. A 440 bp fragment of the human C-reactive protein gene was amplified as positive control.

 

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 40 cycles.

 

 

Recommendations

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 chloride (+ 0.5 - 1.5 mM) might be needed in case of longer amplicons >500 bp.

Direct Oral Swab 2x PCR Master Mix can also be used for real-time cycling, when using fluorescence-based hydrolysis probes. 

 

Composition / Content of the Product

Direct Oral Swab 2x PCR Master Mix contains all components necessary for direct PCR or PCR based genotyping directly from saliva or buccal swab samples including an engineered DNA polymerase, an specifically optimized reaction buffer system, and ultrapure dNTPs. An aptamer-based hot-start formulation of the included DNA polymerase prevents false amplification. Temperatures above 50°-55°C cause the aptamer’s secondary structure to melt and will set-free the polymerase. Only target specific primers and samples need to be added.

 

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: Direct Oral Swab 2x PCR Master Mix was tested for successful allele-specific PCR performance detecting a genomic SNP (rs72921001) in human genomic DNA from saliva samples. 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 40 cycles.

DNA polymerase activity: HiDiTaq 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 Direct Oral Swab 2x PCR Master Mix in probe-based assays?

Yes, HiDiTaq DNA polymerase has a 5'-3'-nuclease activity, so you can use Direct Oral Swab 2x PCR Master Mix in probe-based assays.

What is the error-rate of HiDiTaq 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 Direct Oral Swab 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?

HiDiTaq 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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