RNase H-dependent PCR rhPCR: improved specificity and

rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification, These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection,

RNase H-dependent PCR rhPCR: improved specificity and

Application of rhPCR to determine genotype for 31 individuals at the SMAD7 rs4939827 SNP Locus, Human DNA samples from 31 individuals comprising 10 C/C homozygotes circles, 10 T/T homozygotes squares, and 11 C/T heterozygotes triangles were obtained from the Coriell Cell Repository and genotypes were determined using rhPCR, Reactions were run in triplicate using 2 ng …

rhPCR primers

rhPCR is a novel nucleic acid amplification technology that provides improved accuracy over traditional PCR, rhPCR primers rhPrimers, unique primers that contain RNA bases, are used in conjunction with the thermostable RNase H2 enzyme to perform rhPCR, Reduce the formation of primer-dimers or misprimed PCR products ; Multiplex DNA amplification more accurately, even for a high number of

RNase H2 Enzyme

rhPCR virtually eliminates primer-dimer artifacts, rhPCR uses activation of RNA-DNA hybrid primers by the RNase H2 enzyme from Pyrococcus abyssi see Technology overview section, The primers are only activated upon precise annealing to the correct target, and this RNase H2 activation mechanism nearly eliminates primer-dimers and other amplification artifacts that can affect experimental results,

RNase H-Dependent PCR

The rhPCR primers are designed to contain an RNA residue and are modified usually with a C3 spacer at or near the 3´ end of the oligonucleotide to “block” extension by DNA polymerase, During the annealing step of rhPCR, RNase H2 recognizes and cleaves the RNA:DNA base pair in the primer:target hybrids, Cleavage occurs at the 5´ end of

RNase H-dependent amplification improves the accuracy of

rhPCR technique improves the specificity of RCA-LAMP, To suppress non-specific amplification, we utilized rhPCR technique, For this, RNase H2 enzyme and rh primer with blocking modification at the 3′ end and 6th DNA from the 3′ end replaced with RNA are used, RNase H2 cleaves the replaced RNA only when its periphery forms a complete complementary strand, The blocking …

A High-Performing and Cost-Effective SNP Genotyping Method

We have developed a novel dual enzyme chemistry called rhAmp® SNP genotyping based on RNase H2-dependent PCR rhPCR that provides high signal and specificity for SNP analysis, rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci, Activation of the blocked primers occurs upon hybridization to its perfectly …

Targeted TCR Amplification from Single-Cell cDNA Libraries

Single-cell sequencing of TCR alleles enables determination of T cell specificity, Here we describe a sensitive protocol for targeted amplification of TCR CDR3 regions from single-cell full-length cDNA libraries, By exploiting the specificity of RNase H-dependent PCR rhPCR, the protocol achieves a …

Most Plasmodiophora brassicae Populations in Single Canola

The rhPCR program consisted of initial denaturation at 94°C for 3 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 45 s, and extension at 72°C for 1 min; and a final extension at 72°C for 5 min, For specific amplification of DNA fragments from the genomic DNA of single clubroot galls, rhPCR was conducted in a 100-µl reaction mixture containing 50 ng of

Tracking sample identity using multiplexed rhPCR and

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Tracking sample identity using multiplexed rhPCR and amplicon sequencing in NGS workflows Introduction Katharine Dilger 1, Yongming Sun , Sue Rupp2, Wendy Lee 1, Kevin Lai , Nicole Sponer1, Lisa Bogh2, Madelyn Light1, Scott Rose2, Yu Wang 1, and Yun Bao * 1 Integrated DNA Technologies, Redwood City, CA 2 Integrated DNA Technologies, Coralville, IA

A High-Performing and Cost-Effective SNP Genotyping Method

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SNP genotyping chemistry include enhanced allelic discrimination by rhPCR, and high signal generation and cost reduction by the universal reporter system, 2, Materials and Methods 2,1, SNPs, Genomic DNA Samples and Extraction SNP targets: A total of 130 SNPs were randomly selected from dbSNP Build 144

MIGUEL ALCAIDE

MIGUEL ALCAIDE , Lund, Skåne, Sverige , Senior Molecular Diagnostics Scientist, dPCR at SAGA Diagnostics AB , I have accumulated more than ten years of experience regarding molecular biology techniques and becoming an expert concerning the development and optimization of molecular methods to address biological questions during my scientific career,

DNA Sequence Dimorphisms in Populations of the Clubroot

The rhPCR protocols developed in this study, for the first time, provide a tool for the differentiation between the tested pathotypes, More importantly, the same rhPCR principle can be used in quantitative PCR qPCR, Nucleotide dimorphisms present on all four genes investigated in this study and likely on other polymorphic genes allowed the design of rhPCR primers that can be used for …

Frontiers

Exon-spanning rhPCR primers rhPrimers were designed for each of our 14 pancreatic gene targets, Each rhPrimers contains a single RNA base and are used in conjunction with thermostable RNase H2 enzyme to perform rhPCR, When rhPrimer anneal to the intended pancreatic specific target cDNA, a substrate for RNaseH2 is formed at the site where the single RNA residue resides in the primer, When