Setter, Robert .
Novel Molecular Tools from IDT for Plant Genotyping and Rare Allele Detection.
rhPCR: A Novel Technique for Enhancing Specificity and/or Sensitivity of PCR Type II Ribonuclease H (RNase H2) enzymes cleave nucleic acid substrates on the 5’ side of an RNA located immediately 3’ of a DNA base, resulting in products with a 3’ hydroxyl and 5’ phosphate. The RNase H2 enzyme from the hyperthermophile Pyrococcus abyssi (P.a.) was utilized to perform PCR with primers containing a single ribonucleotide residue and a 3’ end-block. Removal of this block is carried out by the RNase H2 enzyme, and allows these primers to become active (rhPCR). As the primer must first hybridize with the target sequence to have the block removed, the formation of primer-dimers and mis-amplification of closely related sequences is either eliminated, or greatly reduced. Mismatches near the scissile linkage reduce the efficiency of cleavage by P.a. RNase H2, increasing the specificity of the assay. When detecting single nucleotide polymorphisms (SNPs), rhPCR showed more sensitivity than standard allele-specific PCR. The best discrimination was seen when the mismatch was placed at the RNA:DNA base pair, due to the repeated interrogation of the SNP at this position. rhPCR can be used for rare-allele detection and it is possible to identify a mutant allele in a background of 10,000-fold excess of the wild type sequence. Improved Performance of Targeted Capture Exome Sequencing Using Lockdown® Probes Plant and mammalian genomes are large, often comprising greater than 3 billion bases of DNA. However, only a small portion (1-2%) of this sequence is actually translated into proteins. Sequencing the entire genome is costly in both time and resources. Capture-enrichment methods allow focused sequencing of specific areas of interest within the genome, such as the protein-encoding exome or focused subsets of the exome. Integrated DNA Technologies’ (IDT’s) xGen® Lockdown® Probes have been successfully used for both research and medical resequencing applications in the human genome with great success. These probes are 5′ biotin modified oligonucleotides, manufactured using IDT Ultramer® synthesis technology to ensure more full-length product. A size range of 108–120 bp provides optimal coverage while avoiding repeat regions and homologous sequences. xGen Lockdown Probes are individually synthesized, and analyzed by electrospray ionization mass spectrometry before pooling, ensuring that each probe is equally represented in the xGen Lockdown Exome Panel. Even probes with high GC and AT content can be synthesized. Probes that do not pass quality control are documented and resynthesized, providing both premade and custom probe panels a unique advantage over array-derived pools, in which missing or defective probes cannot be identified before sequencing. xGen Custom Lockdown probes are a powerful tool for use in plant genotyping and targeted resequencing applications. Our Speaker Dr. Joseph Dobosy is a Senior Research Scientist at Integrated DNA Technologies. For the past seven years, he has been working to develop the RNase H2-dependant PCR system (rhPCR), a novel technique that enhances specificity of PCR by coupling it to RNase H2. Before joining IDT, Dr. Dobosy did his post-doctoral research at the University of Wisconsin, Madison on the epigenetics of Prostate Cancer. He obtained his doctorate from the University of Oregon in 2003 investigating the connections between DNA methylation and histone acetylation in fungi.
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1 - Integrated DNA Technologies
Presentation Type: Workshop
Location: Salon 13/The Shaw Conference Centre
Date: Sunday, July 26th, 2015
Time: 10:00 AM
Candidate for Awards:None