United States Patent (19)
`Coull et al.
`
`54
`
`(75)
`
`METHODS FOR SUPPRESSING THE
`BINDING OF DETECTABLE PROBESTO
`NON-TARGET SEQUENCES IN
`HYBRIDIZATIONASSAYS
`
`Inventors: James M. Coull, Westford; Jens J.
`Hyldig-Nielsen, Holliston, both of
`Mass.; Sven E. Godtfredsen, Vaerlose,
`Denmark; Mark J. Fiandaca, Acton;
`Kyriaki Stefano, Hopkinton, both of
`Mass.
`
`Assignees: Boston Probes, Inc., Bedford, Mass.;
`Dako A/S, Glostrup, Denmark
`
`Appl. No.: 08/963,472
`Filed:
`Nov. 3, 1997
`Related U.S. Application Data
`
`Continuation-in-part of application No. 08/937,709, Sep. 25,
`1997, abandoned.
`Provisional application No. 60/032,349, Dec. 4, 1996.
`Int. Cl." ............................ C12O 1/68; C12P 19/34;
`C07H 21/02; CO7H 21/04
`U.S. Cl. .................................... 435/6; 435/5; 435/7.1;
`435/91.1; 435/91.2; 530/300, 530/350;
`530/387.1; 530/388.1; 536/23.1; 536/24.3;
`536/24.31; 536/24.32; 536/24.33
`Field of Search ........................... 435/5, 6, 7.1, 91.1,
`435/91.2; 530/387.1, 388.1, 300, 350; 536/24.3,
`24.31, 24.32, 24.33, 23.1
`
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`USOO6110676A
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`(45) Date of Patent:
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`Primary Examiner Jeffrey Fredman
`Attorney, Agent, or Firm Brian D. Gildea
`57
`ABSTRACT
`This invention relates to methods, kits and compositions
`Suitable for the improved detection, analysis and quantita
`tion of nucleic acid target Sequences using probe based
`hybridization assays. The invention is more specifically
`directed to methods, kits and compositions Suitable for
`Suppressing the binding of detectable nucleic acid probes or
`detectable PNA probes to non-target nucleic acid Sequences
`in an assay for a target nucleic acid Sequence to thereby
`improve the reliability, Sensitivity and Specificity of the
`assay. The methods, kits and compositions of this invention
`are particularly well Suited to the detection and analysis of
`nucleic acid point mutations.
`
`41 Claims, 19 Drawing Sheets
`
`MPI EXHIBIT 1064 PAGE 1
`
`MPI EXHIBIT 1064 PAGE 1
`
`

`

`6,110,676
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`U.S. Patent
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`Aug. 29, 2000
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`Sheet 1 of 19
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`6,110,676
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`Figure 1
`
`WY 388
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`RAN' RX 3
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`MPI EXHIBIT 1064 PAGE 6
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`U.S. Patent
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`Aug. 29, 2000
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`Sheet 2 of 19
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`6,110,676
`
`Figure 2
`
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`MPI EXHIBIT 1064 PAGE 7
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`U.S. Patent
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`Aug. 29, 2000
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`Sheet 3 of 19
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`6,110,676
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`Figure 3
`
`Molecular Diagram
`
`
`
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`MPI EXHIBIT 1064 PAGE 8
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`

`

`U.S. Patent
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`Aug. 29, 2000
`
`Sheet 4 of 19
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`6,110,676
`
`Figure 4A
`
`
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`MPI EXHIBIT 1064 PAGE 9
`
`MPI EXHIBIT 1064 PAGE 9
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`MPI EXHIBIT 1064 PAGE 9
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`

`

`U.S. Patent
`
`Aug. 29, 2000
`
`Sheet S of 19
`
`6,110,676
`
`Figure 4B
`
`labeled Wild Type Probes
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`MPI EXHIBIT 1064 PAGE 10
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`MPI EXHIBIT 1064 PAGE 10
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`

`

`U.S. Patent
`
`Aug. 29, 2000
`
`Sheet 6 of 19
`
`6,110,676
`
`Figure 4C
`
`
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`MPI EXHIBIT 1064 PAGE 11
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`U.S. Patent
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`Aug. 29, 2000
`
`Sheet 7 of 19
`
`6,110,676
`
`Figure 4D
`
`Match to Mismatch Ratio
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`MPI EXHIBIT 1064 PAGE 12
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`MPI EXHIBIT 1064 PAGE 12
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`

`

`U.S. Patent
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`Aug. 29, 2000
`
`Sheet 8 of 19
`
`6,110,676
`
`Figure 5A
`
`
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