Chimeric Oligonucleotides for Ligation-Enhanced Nucleic Acid Detection, Methods and Compositions Therefor

a technology of chimeric oligonucleotides and nucleic acid detection, applied in the field of compositions, methods, kits for detecting and/or quantifying nucleic acid molecules in samples, can solve the problems of inability to amplify longer products, inability to reliably detect genomic and gene expression data, inaccurate measurements, etc., and achieve the effect of ligation-enhanced nucleic acid detection

Inactive Publication Date: 2012-03-15
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, modifications of DNA and RNA due to degradation or sample treatment tend to inhibit the ability of traditional polymerases to replicate template sequences, often resulting in inaccurate measurements and unreliable genomic and gene expression data.
Certain of the environments may result in compromised samples.
When the RNA quality is poor, the ability to amplify longer products decreases.

Method used

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  • Chimeric Oligonucleotides for Ligation-Enhanced Nucleic Acid Detection, Methods and Compositions Therefor
  • Chimeric Oligonucleotides for Ligation-Enhanced Nucleic Acid Detection, Methods and Compositions Therefor
  • Chimeric Oligonucleotides for Ligation-Enhanced Nucleic Acid Detection, Methods and Compositions Therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

Archived FFPE RNA is Fragmented

[0242]Samples were obtained from 1) frozen kidney tissue, 2) ambient-stored FFPE lung tissue blocks, and 3) ambient-stored FFPE colon tissue blocks. RNA from approximately 10 μm slices of the tissue samples was extracted and purified using the RECOVERALL® kit (Ambion Inc., Austin, Tex.). Total RNA from each sample (about 100 ng to 150 ng) was analyzed on an AGILENT® 2100 bioanalyzer (AGILENT® Technologies, Santa Clara, Calif.) using default parameters. A typical level of detection for the AGILENT® bioanalyzer is about 0.1 ng / μl.

[0243]An analysis of RNA recovered from frozen kidney tissue is provided by FIG. 6A. An analysis of RNA recovered from ambient-stored FFPE lung tissue blocks and ambient-stored FFPE colon tissue block samples is provided by FIG. 6B and FIG. 6C, respectively. For the figures, the y-axis shows fluorescence intensity in relative units generated by dye binding to RNA, thus peak height is proportional to the amount of RNA. The x-axis...

example 2

Ligation-Enhanced Nucleic Acid Detection Assay Protocol

[0245]The following Table 1 and Table 2 are referred to throughout the following examples.

TABLE 1RNA Dilution Buffer80%Deionized formamide300 mMSodium acetate pH 6.4100 mMSodium citrate pH 6.4 1 mMEDTA 5%PEG 8000100 ng / mlE.coli RNA2× Hybridization Buffer20%Deionized formamide300 mMSodium acetate pH 6.4100 mMSodium citrate pH 6.4 1 mMEDTA 5%PEG 800010× Ligase Buffer500 mMTris pH 7.0100 mMMgCl2100 mMDTT 10 mMATPRNase Cocktail (AMBION ® Inc., Austin, TX #2286)0.5 U / μlRNase A 20 U / μlRNase T110× Complete (SUPERTAQ ™, AMBION ® Inc., Austin, TX) Buffer100 mMTris pH 9.0500 mMKCl 15 mMMgCl2

TABLE 21.Total RNA sample is serially diluted with warmed (37° C.) RNA Dilution Buffer to a range of final concentrations (20 ng / μl, 2 ng / μl, 0.2 ng / μl and 0.02 ng / μl); each sample is heated to 65° C. for 5 minutes and then placed on ice.2.A set of chimeric oligonucleotide probes are mixed and diluted to 50 fmol / μl (50 nM) each in nuclease-free water.3...

example 3

[0246]Standard qRT-PCR vs. Ligation-Enhanced Nucleic Acid Detection for β-Actin mRNA in Archived Tissue

[0247]RNA from the 2-yr old and 13-yr old FFPE colon tissue samples depicted by FIG. 8A and FIG. 8B was used to compare standard qRT-PCR to ligation-enhanced nucleic acid detection embodiments for limits of detection of the β-actin mRNA target nucleic acid. RNA from approximately 10 μm slices of the tissue blocks was extracted and purified using the RECOVERALL® kit (Ambion Inc., Austin, Tex.). Total RNA from each sample was serially diluted with RNA Dilution Buffer (Table 1).

[0248]This study of standard qRT-PCR versus embodiments of ligation-enhanced nucleic acid detection essentially compares the ability of PCR to amplify the “target region” of a target nucleic acid using primers that have sequence complementarity to sequences that are upstream and downstream of that “target region” with the ability of PCR to amplify the “target region” as represented by a ligated product and usin...

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Abstract

Ligation-enhanced nucleic acid detection assay embodiments for detection of RNA or DNA are described. The assay embodiments rely on ligation of chimeric oligonucleotide probes to generate a template for amplification and detection. The assay embodiments are substantially independent of the fidelity of a polymerase for copying compromised nucleic acid. Very little background amplification is observed and as few as 1000 copies of target nucleic acid can be detected. Method embodiments are particularly adept for detection of RNA from compromised samples such as formalin-fixed and paraffin-embedded samples. Heavily degraded and cross-linked nucleic acids of compromised samples, in which classic quantitative real time PCR assays typically fail to adequately amplify signal, can be reliably detected and quantified.

Description

[0001]The present application is a divisional of U.S. patent application Ser. No. 12 / 147,847 filed Jun. 27, 2008, which is a non-provisional of, and claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 60 / 946,624 filed Jun. 27, 2007; the contents of which are incorporated herein in their entirety. The U.S. government has certain rights in this application pursuant to grant no. PHS 2004-2 from the National Institutes of Health and SB grant no. R43 GM075511.FIELD[0002]The present teachings generally relate to compositions, methods, and kits for detecting and / or quantifying nucleic acid molecules in a sample.INTRODUCTION[0003]Real-time PCR is routinely used for detection of nucleic acid, and real-time quantitative reverse transcriptase-PCR (qRT-PCR) is routinely used for detection of RNA and for studying gene expression. However, modifications of DNA and RNA due to degradation or sample treatment tend to inhibit the ability of traditional polymerases to replic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/06
CPCC12Q1/6816C12Q1/6827C12Q2561/125C12Q2525/161C12Q2525/155C12Q1/6851C12Q1/6855
Inventor KUERSTEN, R. SCOTTPASLOSKE, BRITTAN L.
Owner LIFE TECH CORP
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