DNA / PCR

DirectBlood Genotyping PCR Kit

allows genotyping without any previous DNA isolation. Save time and money: directly use EDTA blood without any extraction steps.

The DirectBlood Genotyping PCR Kit is optimized to function with a wide range of different SNPs to detect possible nucleotide variations.  It has been successfully tested with a variety of hydrolysis probes and hybridization probes.

Ship and store the DirectBlood Genotyping PCR Kit simply at room-temperature. A cool chain is not needed anymore. The included master mix is freeze-dried and dissolves within a few seconds after addition of the hydration buffer, add primer/probes and your blood sample and the PCR can be started immediately.

 

Article Product Size Price
#5000S DirectBlood Genotyping Kit

 100 reactions à 20 µl (4 LyoCakes)

€ 199,-
#5000M DirectBlood Genotyping Kit  500 reactions à 20 µl (20 LyoCakes) € 899,-

Bulk offer: Should you require larger amounts (> 1000 reactions), significant discounts are available.

Please contact us to receive an individual quotation.

 

Upon request:

There are already SNP-assays available. Please provide us with the rs-number of the SNP-assays your are interested in to receive an individual quotation.

Article Product Size Price
#5100S DirectBlood Genotyping Kit including specific SNP assay primer/probe mix (4x)

100 reactions à 20 µl

please request
#5100M DirectBlood Genotyping Kit including specific SNP assay primer/probe mix (4x) 500 reactions à 20 µl please request

Upon request, customized freeze-dried DirectBlood Genotyping PCR mixes, which are already containing (your) SNP-specific primers&probes can be provided as well. Please contact us to receive an individual quotation.

 

download-pdf Product manual (PDF) Order

 

 

Applications

Applications

  • Direct PCR from blood - without DNA extraction
  • Genotyping & genomic profiling
  • End-point PCR
  • Real-time PCR with with fluorescence-based hydrolysis probes

Reaction Setup

Preparations before use

Please rehydrate the DirectBlood Genotyping Lyocake by adding exactly 218 µl of the respective Rehydration Buffer to the PCR mix, resulting in 250 µl of ready-to-use 2x Master Mix. Subsequently invert the closed tube a few ties or briefly vortex and spin down the mixture before use. Tube should be placed on ice after rehydration.

The rehydrated 2x DirectBlood Genotyping PCR Mix is now ready to be used for setting up a PCR experiment or can be stored at -20°C.

 

Sample preparation

Dilute a given EDTA blood sample to 2% (v/v) in DNase-free water by adding of 10 µL blood to 490 µL water. Subsequently invert the closed tube a few times or briefly vortex the mixture before use. The in this way prepared sample is ready to be used for Genotyping PCR and can also be stored for a couple of weeks at 4°C, e.g., for re-testing purposes.

 

Recommendation for sample handling

  • Spin down and mix all solutions carefully before use.
  • Include a control without sample as a negative control.
  • Minimize the number of freeze-thaw cycles by storing the rehydrated product in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C.
  • We recommend the use of disposable gloves, DNase and RNase free filter tips and plastics.

 

PCR Reaction setup

Component Volume Final concentration
2x DirectBlood Genotyping PCR Mix 10 µl 1x
4 x Primer/Probe Mix  5 µl 1x
Diluted blood sample (2%) 5 µl 0.5%
Total volume/reaction 20 µl

Keep all components on ice.
Spin down and mix all solutions carefully before use.

Example Results

Example Results:

DirectBlood Genotyping Kit (#5000) hydrolysis probe assay

The DirectBlood Genotyping Kit is compatible with a wide range of assays. Here we are detecting a SNP in the Factor V gene (rs6025; G1691A), which is connected with a higher risk for venous thrombosis, with a fluorescent hydrolysis probe assay.

Homozygous wild type blood samples will show a strong signal and amplification plot (blue curve) in channel 2 (Cy5) and none or a very low signal

(red curve) in the other channel 1 (ROX).

 

 

Heterozygous blood samples will give an intermediate signal and amplification plots with very similar intensities in both channels 1 and 2 (blue and red curve).
 
 
 
 
Homozygous mutant blood samples, channel 1 (ROX) will show a strong signal and amplification plot (red curve) and channel 2 (Cy5) none or a very low signal and amplification plot (blue curve). So the signals are reversed on homozygous mutant samples.  
 
 
 

 

Example Results:

DirectBlood Genotyping Kit (#5000) hybridization probe assay

Here we are detecting the same SNP in the Factor V gene (rs6025; G1691A), which is connected with a higher risk for venous thrombosis, with a fluorescent hybridization probe assay.

Three blood samples with known genotype were used. All the genotypes can clearly be identified. The blue curve shows the specific wild type (homozygous G;G) peak at 61°C. The red curve shows the specific mutant (homozygous A;A) peak at 53°C. A heterozygous case (A;G) will result in both melting curve peaks, respectively at the same specific temperature for wild type and mutant (orange curve).

 

PCR protocols

Typical PCR protocol (hydrolysis probe protocol)

Step Temperature Time
Initial denaturation 95°C 2 min
Denaturation 95°C 10 sec
Annealing/Extension* 60°C 40 sec   (50 cycles) 

* Fluorescence data should be collected during this step. Real-time PCR signals may be reduced to 25% of the signals obtained from extracted DNA samples. This is a normal result and will still be leading to significant, valid Ct-values and unambiguous melting peaks.

Please note: Suggested cycling protocols can vary and depend strongly on the cycler and applied PCR assay.

 

 

Composition / Content of the product

DirectBlood Genotyping PCR Kit contains all components necessary for direct PCR or PCR based genotyping directly from blood 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 probes need to be added.

 

Quality Control Assay

DirectBlood Genotyping PCR Kit is tested for functional and accurate genotyping PCRs direct from EDTA blood samples.
DNA polymerase activity: DNA polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and 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.

 

Storage

The freeze-dried DirectBlood Genotyping PCR Lyocake can be stored at room-temperature. Please store resuspended master mixes (2x) and primer/probe mixes (4x) 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. Please store the included rehydration buffer and additional primer/probe mixes upon arrival at -20°C.

 

 

img-faqsFAQ`s

Can I pre-mix the 2x PCR mix with the 4x primer/probe mix before start?

Yes you can already combine the 2x DirectBlood Genotyping PCR Mix with the 4 x Primer/Probe Mix. Freshly prepare this before each PCR run, as we recommend to keep this mix on ice and to use it up within 1 hour.

Which PCR platform do you recommend to use?

The assays are established on a Lightcycler 480 and a Quanstudio 5 system. However, a successful PCR setup can likely be found with another PCR cycler as well. Most important is that the real-time cycler supports the mentioned fluorescent channels for the detection.

How long can I store the diluted blood samples before PCR?

In our tests, we were still able to reliably detect genomic alleles after storing dliuted EDTA blood samples in water (2%) at 4°C for at least 3 weeks.

 

 


 

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