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Highly orthogonal universal sequences for use in nucleic acid assays

A general-sequence, highly advanced technology that can be used in the determination/examination of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc. It can solve the problems of analyte cross-reactions that are difficult to overcome and eliminate, and reduce the effectiveness of multiple bDNA analysis.

Inactive Publication Date: 2008-01-23
SIEMENS HEALTHCARE DIAGNOSTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hybridization of a natural base to any of its three mismatched bases can significantly reduce the validity of multiplexed bDNA analysis since only one pairing is successful for each natural base
Additionally, with the development of complex multiplex assays, unwanted cross-reactivity between analytes is difficult to overcome and eliminate

Method used

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  • Highly orthogonal universal sequences for use in nucleic acid assays
  • Highly orthogonal universal sequences for use in nucleic acid assays
  • Highly orthogonal universal sequences for use in nucleic acid assays

Examples

Experimental program
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Embodiment 1

[0086] The practice of the present invention may employ, unless indicated to the contrary, conventional techniques of synthetic organic chemistry, biochemistry, molecular biology and the like, as known in the art. These techniques are fully described in the literature, see Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); OLIGONUCLEOTIDE SYNTHESIS (M.J.Gait, ed., 1984); THE PRACTICE OF PEPTIDE SYNTHESIS (M.Bodanszky and A .Bodanszky, 2nd ed., Springer-Verlag, New York, NY, 1994); NUCLEIC ACID HYBRIDIZATION (B.D. Haines & S.J. Higgins, eds., 1984); and METHODS IN ENZYMOLOGY (Academic Press, Inc.).

[0087] In the following examples, it is to be understood that while efforts have been made to ensure accuracy of experimental parameters (eg, amounts, temperature, etc.), some experimental errors and deviations should be accounted for in repeating the experiments described below. Unless indicated to the contrary, temperature values ​​are in degrees Celsiu...

Embodiment 2

Generation of Best Orthogonal Utility Sequence

[0094] Multiple optimal CPs were designed from a 20-mer sequence containing a hexabase codon consisting of four natural bases and two unnatural bases, iso-G and iso-C. Figure 1A shows the general structure of the starting 20-mer sequence of the present invention, which consists of four natural bases in no particular order separated by an iso-G or iso-C base. To generate probes from the starting 20-mer sequence, the starting sequence was screened by the method described in Example 1 with the aim of generating 33 CPs and 33 CEs. The resulting sequences were used to design a six-plex cytokine analysis panel. Since the probes showed some cross-reactivity in cytokine assays, the probes were further optimized for minimal cross-reactivity by limiting the melting temperature of the probes to about 85°C. The optimal set of probes resulting from screening and testing is described by the formula in FIG. 1B and shown in Table 1. When the...

Embodiment 3

Specificity of Multiplexed Cytokine mRNA Analysis

[0095] The universal orthogonal sequences CP1-CP9 and CE1-CE9 (SEQ ID NO. 1-22) of Table 1 were used for multiplex cytokine mRNA quantification using bDNA design principles and Luminex(R) suspension array technology for readout. Table 2 shows the performance of multiplex mRNA profiling using CP1-CP9 and CE1-CE9 for a target of approximately 60,000,000 molecules for nine cytokines. In this experiment, complete bDNA analysis was performed for each cytokine. As shown in Table 2, a very strong RFU signal was observed on the expected bead and there was negligible cross-hybridization of the selected universal sequences compared to the background signal of the assay bead. Figure 2 shows the results of this analysis in a three-dimensional histogram.

Table 2

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Abstract

The invention provides a set of highly orthogonal six-code universal sequences for use in bDNA singleplex and multiplex nucleic acid hybridization assays. The six-code orthogonal sequences do not cross-hybridize and thus, minimize or eliminate the 3-mer cross-hybridization inherent in the second and third generation bDNA assays. The highly orthogonal universal sequences may be used in singleplex or multiplex bDNA assays quantitatively and qualitatively to determine mRNA levels in a sample; to screen for and genotype targets, such as viruses, that are present in low volumes in a sample; to screen for and genotype SNPs; and to measure changes in the amount of a gene in a sample such as when gene amplifications or deletions occur. The highly orthogonal universal sequences may also be used as universal capture probes to selectively bind assay components in a way that facilitates their further analysis.

Description

technical field [0001] In general, the invention relates to the generation of universal sequences for nucleic acid analysis. More specifically, the present invention relates to the generation of highly orthogonal universal sequences with negligible cross-reactivity for analysis of branched DNA ("bDNA") and other nucleic acids. Background technique [0002] The bDNA assay, developed by Chiron Diagnostics and now owned by Bayer Diagnostics, uses linear rather than exponential signal amplification to increase the sensitivity and specificity of quantitative hybridization in diagnostic tests. Collins et al, NUCLEIC ACID RESEARCH 25(15):2979-2984 (1997). bDNA analysis is used to quantify RNA and DNA targets from a variety of sources. The sensitivity and specificity of the assay derives in part from judicious selection of the oligonucleotide probes that make up the probe set. [0003] In a bDNA assay, a capture probe (“CP”) is attached to a solid support and a capture assist (“C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q2525/101Y02A50/30
Inventor M·郑D·阿尔B·沃纳M·吴C·-A·常
Owner SIEMENS HEALTHCARE DIAGNOSTICS INC
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