Overcoming Challenges in Multiplex PCR Analysis

Fast and easy multiplex PCR without optimization
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Multiplex PCR is a challenging technique that requires amplification of two or more products in parallel in a single reaction tube. This technology was first described by Chamberlain et al. in 1988, and since then has been applied in many areas of DNA testing, including analyses of deletions, mutations, and polymorphisms, or quantitative assays.

Two important challenges associated with multiplex PCR technologies include laborious, time-consuming optimization procedures and unsuitability of traditional separation technologies (such as agarose slab gels) for separation of multiplex PCR products, especially if these products only differ by a few base pairs.

This article addresses these challenges and presents innovative solutions that provide easy and sensitive multiplex PCR and superior DNA fragment analysis in as little as 10 minutes.
Fast and easy multiplex PCR without optimization
Effortless DNA fragment analysis
Fast and easy multiplex PCR without optimization
Multiplex PCR is a highly demanding technique used for genotyping applications such as STR analysis or mutation detection and can be used to amplify and detect even a single copy of a nucleic acid sequence. However, amplification of such a low number of target sequences in parallel with more abundant sequences is often limited by the generation of nonspecific PCR products and primer–dimers or by the amount and quality of template DNA. Traditionally, multiplex PCR has required extensive optimization of annealing conditions, and enzyme and buffer concentrations for maximum amplification efficiency of each target. In addition, a stringent hot-start procedure and specially optimized buffer systems are absolutely crucial for successful results. Compared to standard PCR systems using 2 primers, an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs. Primers that bind with high efficiency utilize more of the PCR reaction components, thereby reducing the yield of other PCR products. This often results in unamplified DNA sequences and missing bands when run on an agarose gels.

QIAGEN has overcome this problem with the specially adapted QIAGEN Multiplex PCR Buffer (provided with the QIAGEN Multiplex PCR Kit). This buffer contains a unique synthetic additive, Factor MP, which further promotes stable and efficient annealing of several different primers to the nucleic acid template (see figure Factor MP promotes stable and efficient annealing). The increased hybridization efficiency and primer stability provide excellent product yields — even for primer pairs that bind suboptimally to their target sequence under the chosen conditions. Therefore, the QIAGEN multiplex PCR reagent allows most primer–template systems to be combined in a single reaction without optimization, making it highly suitable for all genotyping applications (see figure Easy high-resolution analysis of 15-plex PCR). The master mix format offers convenience and ease of use. Pre-optimized concentrations of HotStarTaq DNA Polymerase ensure high specificity and sensitivity. Nonspecific annealing is minimized, which leads to maximal yields of specific PCR products (see figure Unique PCR buffer increases specific primer annealing).

Effortless DNA fragment analysis
Currently, the most commonly used method for nucleic acid separation is gel electrophoresis, using manually poured slab gels. However, this method is highly labor-intensive and exposes users to hazardous chemicals such as ethidium bromide. In addition, thorough analysis of the data in terms of fragment sizes and concentration can be tedious – especially when the data is to be compared with previously analyzed PCR products. Standardization is of key importance when comparing data from different gel runs and this places a great emphasis on accurate electrophoresis conditions and record keeping.

Analysis of multiplex PCR products can be made easier, faster, and more standardized using the QIAxcel Advanced system (see figure QIAxcel Advanced System), which fully automates high-resolution capillary electrophoresis of up to 96 samples without manual intervention. It also ensures greater safety as exposure to hazardous chemicals such as ethidium bromide is minimized. The high detection sensitivity provided by the QIAxcel Advanced analyzer enables robust results even with low concentrations of nucleic acid (see figure Easy high-resolution analysis of 15-plex PCR). Standardization is achieved through the use of pre-cast gel cartridges that allow up to 96 samples to be run using precisely defined electrophoresis conditions. Results can be viewed in real-time and the data can be easily stored and analyzed allowing simple comparison of data generated from different sample runs. Therefore, the QIAxcel Advanced System speeds up nucleic acid analysis applications by eliminating slab-gel analysis while streamlining entire sample purification and analysis workflow.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

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High-resolution analysis of multiplex PCR samples
Easy and high-resolution analysis of 15-plex multiplex PCRs.
PCR products were generated using the QIAGEN Multiplex PCR Kit according to the standard protocol. PCR samples (13 μl) were analyzed on [A] a 2% agarose gel or [B] using the QIAxcel system with the QIAxcel DNA High Resolution Cartridge and the preinstalled OM500 method. The gel image produced by the QIAxcel system shows much higher resolution than the standard agarose gel technique. [C] Each sample lane can be visualized individually in electropherogram form. Lane 7 is shown.
NH4+ and K+ cations in QIAGEN PCR buffers increase specific primer annealing
Unique PCR buffer increases 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 (B), destabilizing principally 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.
Unique Type-it Microsatellite PCR Buffer Promotes Stable and Efficient Annealing
Factor MP promotes stable and efficient annealing.
[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.
QIAxcel Advanced System.