PCR assays are the most rapid, most sensitive, and least expen- sive way to assess the abundance of mutant DNA fragments present in liquid biopsies. “SuperSelective” PCR primers, due to their unique design, are extraordinarily specific, able to selectively initiate the synthesis of amplicons on ten mutant DNA fragments in the presence of 1,000,000 wild-type DNA fragments. Sets of SuperSe- lective primers, each possessing unique 5′-tag sequences, enable the amplicons generated from each mutant to be distinguished by differently colored molecular beacon probes. And the inclusion of primers for a wild-type reference gene fragment, enables the abundance of each type of mutant DNA fragment to be assessed by determining the difference between its threshold value and the threshold value of the reference gene.
Mikael Kubista 1, Amin Forootan2
1 TATAA Biocenter AB, Göteborg, Sweden
2 MultiD Analyses AB, Göteborg, Sweden
GenEx was the first commercial software developed for qPCR, and has since then become market leader with few thousands of users and several thousands of publications using GenEx. Sup- ported by all leading qPCR instrument manufacturers GenEx has batch import and corrects for inter-run variation. During pre- processing missing values are imputed, outliers are handled, and data are optimally normalized. Absolute quantification includes determining limit of detection, limit of quantification and dynamic range. All parameters are determined with confidence ranges in line with CLSI, CEN and ISO guidelines. Expression data are ana- lyzed with univariate methods including t-test, non-parametric methods and various ANOVAs with appropriate corrections for multiple testing ambiguity or multivariate methods including hierarchical clustering, Principal Component Analysis (PCA), Self- Organizing Map (SOM), Artificial Neural Network (ANN), Support Vector Machines (SVM), and many more. All analyses are easily per- formed with few clicks with the mouse using intuitive workflows that generate results that are readily interpreted in a biologi- cal context. At this lunch seminar, we will present GenEx and exemplify how easily multiple run files are in imported, measured data pre-processed, and how data are interactively analyzed using dynamic PCA to reveal key expression pathways and correlations across samples. All participants will receive FREE GenEx license for limited time.
Download a free GenEx trial version on GenEx.gene-quantification.info
The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. SA Bustin et al., Clinical Chemistry 55 (4), 611-622
rhAmpTM SNP Genotyping is a novel target-activated geno- typing solution utilizing RNase H2-dependent PCR (rhPCR) to provide superior discrimination of single nucleotide polymor- phisms (SNPs), multi-nucleotide polymorphisms (MNPs), and insertion/deletions (InDels). rhPCR genotyping technology com- bines a unique two enzyme system with DNA-RNA hybrid primers to interrogate target SNPs. Allele specific primers contain a 5′ uni- versal tail, a single RNA base targeting the SNP, and a terminal blocking group. The 3′ blocking group is removed and primer acti- vation is achieved only upon hybridization to its perfectly matched target. The thermostable RNase H2 cleaves the primer at the RNA base and releases the blocking group, allowing primer extension. PCR specificity and selectivity is improved with the use of a mutant Taq DNA polymerase and signal generation is achieved using a cost- effective universal reporter system. More than 550 human targets were selected for testing. A proprietary algorithm provided a design rate greater than 95%, and consistently high genotyping perfor- mance was achieved with greater than 90% call rate and 99.5% call accuracy on over 90% of tested assays. The rhAmpTM genotyping solution offers advanced bioinformatics driving a high assay design rate and minimal primer dimer formation, a dual enzyme master mix optimized for selectivity and stability, and an efficient univer- sal reporter system that generates high fluorescent signal, offering increased confidence in SNP calling and the benefit of a lower cost per genotype.
We report here a novel multiplex PCR chemistry called rhPCR. Its primers contain a non-extendable terminal blocker and single RNA base closer to 3′. rhPCR requires both thermostable Type II RNase H (RNase H2) and DNA Polymerase for specific amplification. RNase H2 can activate rhPCR primers by target-specific cleavage of the RNA base while rhPCR primers perfectly bind to the target DNA to form a DNA duplex. Cleaved rhPCR primers can then be extended by a DNA polymerase. Combination of both 3′ blocked rhPCR primers and a highly specific mutant DNA polymerase have eliminated or greatly reduced primer dimers and non-specific amplification, resulting in higher multiplexity and better specificity than standard multiplex PCR. We have demonstrated the feasibility of the multi- plex rhPCR for amplicon sequencing in multiple assay pools ranging from 96- to 1,000-plexes. Major advantages of multiplex rhPCR for amplicon sequencing include better workflow, higher map- pable reads (>97%) and on-target percentage (>97%) comparing to standard multiplex PCR.