QIAGEN Multiplex PCR Kits

Cat No./ID 206143

QIAGEN Multiplex PCR Kit (100)

For 100 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl2, 3 x 0.85 ml), 5x Q-Solution (1 x 2.0 ml), RNase-Free Water (2 x 1.7 ml)

Cat No./ID 206145

QIAGEN Multiplex PCR Kit (1000)

For 1000 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl2, 1 x 25 ml), 5x Q-Solution (1 x 10 ml), RNase-Free Water (1 x 20 ml)

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Cat No./ID 206152

QIAGEN Multiplex PCR Plus Kit (100)

For 100 x 50 μl multiplex PCR reactions: 2x Multiplex PCR Master Mix (3 x 0.85 ml), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml), 10x CoralLoad Dye (1 x 1.2 ml)

Cat No./ID 206152

QIAGEN Multiplex PCR Plus Kit (100)

For 100 x 50 μl multiplex PCR reactions: 2x Multiplex PCR Master Mix (3 x 0.85 ml), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml), 10x CoralLoad Dye (1 x 1.2 ml)

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Multiplex Master Mix

Q-Solution (optional) – amplify difficult/GC-rich templates

  • Facilitates amplification of difficult templates by modifying the melting behavior of DNA
  • Improves suboptimal PCR caused by templates that have a high degree of secondary structure or are GC-rich
  • One working concentration, which has been optimized for multiplex PCR
  • Nontoxic and does not compromise PCR product purity

Amplification of difficult templates with Q-Solution. Two different primer-template systems were amplified in duplicate using QIAGEN PCR Buffer and Taq DNA Polymerase in the absence (-) or presence (+) of 1x Q-Solution. A human angiotensin receptor II gene, B mouse protein kinase C gene. M: markers.

HotStarTaq DNA Polymerase

  • Unique chemical modification of recombinant Taq DNA Polymerase
  • Polymerase activation by initial heat-incubation step
  • Robust activation independent of PCR environment (pH, salts)
  • Prevents extension of nonspecifically annealed primers and primer–dimers

Optimized polymerase concentration

Efficient 19-plex PCR using QIAGEN Multiplex PCR Plus Kit. Multiplex PCR of 19 targets (99–955 bp) was performed using standard conditions for the QIAGEN Multiplex PCR Plus Kit, without further optimization (QIAGEN) or using a variety of concentrations of a hot-start DNA polymerase from Supplier AII.

A Analysis using an agarose gel.

B Analysis using the QIAxcel Advanced System. The QIAGEN Multiplex PCR Plus Kit results in specific amplification of all targets without the need for optimization. Despite lengthy optimization using different enzyme concentrations, multiplex PCR using the kit from Supplier AII resulted in missing fragments, even when using higher concentrations. M: GelPilot 100 bp Plus Ladder.

The unique dual cation buffer

NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing. K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and

as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases, destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer–template binding over a wide temperature range.

Factor MP – higher primer annealing efficiency

  • Factor MP supports macromolecular crowding
    • Displaces H2O at the template
    • Increases the local concentration of primers at the template
  • Leads to more efficient hybridization of primer/probes to the template
  • Supports the binding of Taq DNA polymerase to the primer-template
  • Stabilizes specifically bound primers

A NH4+ ions prevent nonspecific primers from annealing to the template. Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template.

B Together with K+ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.

Experimental design

Target DNA

Challenge 1: High variation in target concentrations and sequence

  • Abundant targets preferred over rare ones
  • Less efficient amplification of GC-rich targets
  • Unreliable results
  • Deviation from singleplex data
QIAGEN multiplex PCR chemistry overcomes this by:
  • Guaranteeing higher primer template binding efficiency with our unique buffer composition and Factor MP
  • Ensuring equal amplification rates regardless of sequence properties using Q-solution

Challenge 2: Amplicon length bias

  • Shorter amplicons preferred over longer amplicons
  • Short fragments (including primer–dimers) compete efficiently for reaction components
  • Nonspecific binding of Taq to newly synthesized short dsDNA
  • Low product yields or missing PCR product
QIAGEN multiplex PCR chemistry overcomes this by:
  • Guaranteeing higher primer template binding efficiency with our unique buffer composition and Factor MP
  • Preventing extension of nonspecifically annealed primers and primer–dimers using HotStarTaq

Primers

Challenges:

  1. Relatively high primer concentration required for efficient primer annealing during short annealing step
  2. Risk of complementary primer sequences
  3. Different hybridization properties of primers
QIAGEN multiplex PCR chemistry overcomes these challenges by:
  • Guaranteeing higher primer template binding efficiency with our unique buffer composition and Factor MP
  • Preventing extension of nonspecifically annealed primers and primer–dimers using HotStarTaq
Recommendations:
  • Always test primer pairs in singleplex PCR first
  • Use software for primer design, such as MPprimer

External links on this web site are provided only for the convenience of QIAGEN web site visitors. QIAGEN has no interest in, responsibility for, or control over non-QIAGEN affiliated linked sites. QIAGEN makes no promises or warranties of any kind, express or implied, including those of merchantability and fitness for a particular purpose, as to content of linked sites. In no event shall QIAGEN be liable for any damages resulting from use of these links even if QIAGEN has been informed of the possibility of such liability.

External links on this web site are provided only for the convenience of QIAGEN web site visitors. QIAGEN has no interest in, responsibility for, or control over non-QIAGEN affiliated linked sites. QIAGEN makes no promises or warranties of any kind, express or implied, including those of merchantability and fitness for a particular purpose, as to content of linked sites. In no event shall QIAGEN be liable for any damages resulting from use of these links even if QIAGEN has been informed of the possibility of such liability.

Sample Preparation

Cat No./ID 51404

QIAamp Fast DNA Tissue Kit

  • Up to 10 times more yield
  • 30 minutes from sample to pure DNA

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Cat No./ID 51104

QIAamp DNA Blood Mini Kits

  • Fast and easy purification of DNA from whole blood
  • Compatible with anticoagulants

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Advantages of Multiplex PCR

  • Co-amplification of internal controls and reference genes
    Increases reliability
  • Conservation of precious samples
    More data per reaction
  • Increased throughput
    More targets in less time
  • Reduced reagent costs
    More results per dollar

Automated PCR analysis with QIAxcel

Automation streamlines your workflow by replacing and overcoming the bottlenecks of traditional slab-gel electrophoresis:

  • Ready-to-run and reusable gel cartridges:
    • Limit error-prone manual steps
    • Bring safety and convenience; no handling of hazardous compounds
    • Save on both time and costs
  • Process up to 96 samples unattended per run
  • Fast processing: 12 samples in 3–10 min
  • High resolution of 3–5 bp up to 500 bp
  • Sample input amounts < 0.1 µl
  • Limit of detection of 0.1 ng/µl
  • Digital data collection ensuring reproducibility and standardization of results

QIAxcel Advanced – automates nucleic acids analysis

High-resolution analysis of multiplex PCR samples. PCR products were generated using the QIAGEN Multiplex PCR Kit according to the standard protocol. PCR samples (13 µl) were analyzed A on a 2% agarose gel and B using using the QIAxcel Advanced system with the QIAxcel DNA High Resolution gel cartridge and the preinstalled OM500 method. B The gel image produced by the QIAxcel Advanced system shows much higher resolution than the agarose gel.