Abscription
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During normal transcription, RNA polymerase transcribes a number of short nonproductive oligonucleotides, and this process is called abortive transcription. The trapped RNAPs have been named abscriptases and the synthesis of specific length oligonucleotides called abscription.[1] Artificial promoters, or abortive promoter cassettes (APCs[2]) are used to make a different abscript of specific length and sequence which can be separated and quantified. Abscription efficiently produces trinucleotides at approximately 1,000 to 10,000 abscripts per minute.
- nGpA-OH + nGTP + APC encoding 5'GpApG + Abscriptase ↔ nGpApG (trinucleotide RNA) + nPPi (inorganic pyrophosphate)
A uniform population of trinucleotides are produced in the presence of a dinucleotide initiator (such as GpA) and a ribonucleoside-triphosphate (such as GTP) encoded by the attached APC. In this example, the attached APC carries the DNA sequence CpTpC and would encode the reiterative syntheis of the ribonucleotide GpApG. A single polymerization step will produce a trinucleotide abscript plus inorganic pyrophosphate. Either or both the trinucleotide or the pyrophosphate produced, or the polymerization step itself, can be detected and quantified. Unmodified abscripts are detected and quantified by LC-MS (liquid chromatography-mass spectrometry or rTLC (rapid thin layer chromatography), but the use of modified nucleotides as abscription substrates allows for other types of detection (capillary electrophoresis, fluorescence).[3] Modifications at the 5’ end of the dinucleotide initiator produces fluorescent abscripts. Attachment of biotin at the 5’ end allows abscript capture.
Molecular biomarkers such as RNA, protein, and DNA (including DNA methylation,[4] amplifications, deletions, insertions, translocations and mutations) can be detected and quantified by covalently attaching an APC to the target.[5][6] Different APCs, encoding different abscripts, can be attached to different targets for multiplex detection. The abscripts produced during abscription are quantified and provide a direct measure of the amount of the biomarker to which an Abortive Promoter Cassette (APC) has been attached.
References[edit]
- ↑ U.S. 7,541,165: Molecular Detection Systems Utilizing Reiterative Oligonucleotide Synthesis; Issued 06/2009; Claims to Abscription methods for detecting proteins
- ↑ U.S. 7,473,775: Abortive Promoter Cassettes; Issued 01/06/2009
- ↑ U.S. Patent 8,211,644 “Molecular beacon based methods for detection of targets by Abscription” Issued 2012
- ↑ David R. McCarthy, Philip D. Cotter, and Michelle M. Hanna (2012). MethylMeter(r): A Quantitative, Sensitive, and Bisulfite-Free Method for Analysis of DNA Methylation, DNA Methylation - From Genomics to Technology, Dr. Tatiana Tatarinova (Ed.), ISBN 978-953-51-0320-2, InTech, DOI: 10.5772/36090. Available from: http://www.intechopen.com/books/dna-methylation-from-genomics-to-technology/methylmeter-a-quantitative-sensititive-and-bisulfite-free-method-for-analysis-of-dna-methylation
- ↑ U.S. 7,470,511: Methods Determining Nucleic Acid Methylation; Issued 12/20/08
- ↑ U.S. 7,468,261: Molecular Detection Systems Utilizing Reiterative Oligonucleotide Synthesis; Issued 12/23/08; Claims to Abscription Methods for Detecting DNA and RNA
Sources[edit]
- U.S. 7,226,738: Molecular Detection Systems Utilizing Reiterative Oligonucleotide Synthesis; Issued 6/05/07; Claims to Abscription methods for detecting multiple reiterated oligonucleotides using a target site probe and/or formation of a bubble complex
- U.S. 7,045,319: Molecular Detection Systems Utilizing Reiterative Oligonucleotide Synthesis; Issued 5/16/06; Claims to Abscription methods for detecting methylated cytosine residues at CpG sites in a target DNA polynucleotide
- U.S. Patent 8,263,339 “Abscription Based Molecular Detection” Issued 2012
- U.S. Patent 8,242,243 “Methods and Reagents for Detecting CpG Methylation with a Methyl CpG Binding Protein (MBP)” Issued 2012
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