Rapid screening method of gene amplification products in polypropylene plates

• US 5,545,528 A
• Filed: 01/26/1995
• Issued: 08/13/1996
• Est. Priority Date: 04/15/1993
• Status: Expired due to Term

• Current Assignee: Hitachi Chemical Research Center, Inc (Resonac Holdings Corp.)
• Original Assignee: Hitachi Chemical Research Center, Inc (Resonac Holdings Corp.)
• Inventors: Mitsuhashi, Masato, Ogura, Mieko
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/379,077
• Time in Patent Office: 565 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 435/91.5, 435/91.52, 435/91.51, 422/55, 422/58, 422/82.08
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence

Abstract

The present invention relates to a method for the rapid screening of gene amplification products on commercially available polypropylene microtiter plates. In one aspect of the invention, polypropylene microtiter plates are used for polymerase chain reaction (PCR) and the amount of nucleic acid sequences amplified through the reaction is quantitated in the same plates. In another aspect of the invention, polypropylene plates are used for detection and quantification of nucleic acids.

Claims

1. A method for rapid screening and analysis of positive gene amplification samples, comprising:

providing a polypropylene reaction vessel having a plurality of wells;

adding a sample containing a nucleic acid having a sequence to be amplified to one or more of said wells;

adding reactants for amplification of said sequence to one or more of said wells containing said nucleic acid;

amplifying said sequence in said sample in said well;

adding a dye to the sample in said well, said dye selectively and quantitatively complexing with the amplified nucleic acid sequence to form a nucleic acid/dye complex, said dye in said complex emitting light energy in response to excitation thereof by light in the visible spectrum, said dye having a DNA partition coefficient in a 10% ethanol/water solution of greater than 1×

10⁷ ;

optically exciting said sample with said light in the visible spectrum;

detecting and measuring light energy emitted from said dye in said complex without transferring said sample from said well in order to determine the presence of amplified nucleic acid sequence in the well.

2. The method of claim 1, wherein the optically exciting step comprises use of a laser.

3. The method of claim 2, wherein the laser emits light having a wavelength of 633 nm.

4. The method of claim 3, wherein said dye is Toto-3.

5. The method of claim 1, wherein the amplifying step is selected from the group consisting of polymerase chain reaction (PCR), self-sustained sequence replication (3SR), ligation amplification reaction system (LAR/LAS) and beta-Q replicase (β

QR).

6. The method of claim 1, additionally comprising layering mineral oil over the reactants in the wells prior to the amplification step.

7. The method of claim 1, wherein the reaction vessel is a microtiter plate.

8. The method of claim 1, wherein the dye has an excitation wavelength of between 400 nm and 700 nm and an emission wavelength of between 400 nm and 700 nm.

9. The method of claim 8, wherein the excitation wavelength is 485 nm and the emission wavelength is 530 nm.

10. The method of claim 8, wherein the dye is selected from the group consisting of Yoyo-1, Toto-1 and Toto-3.

11. The method of claim 1, wherein the volume of the reactants and sample is between about 5 μ

l and about 50 μ

l.

12. The method of claim 11, wherein the volume of the reactants and sample is between about 10 μ

l and 20 μ

l.

13. The method of claim 1, further comprising applying a substance to the exterior of the reaction vessel so as to enhance transmission of the fluorescence.

14. The method of claim 13, wherein said substance comprises mineral oil.

24. The method of claim 6, wherein said oil remains layered over the reactants during the detecting step.

25. The method of claim 1, wherein said dye is a thiazolium or azolium dye.

15. An assay for the detection of a specific nucleic acid sequence in a sample containing nucleic acid from a human subject, comprising:

loading said nucleic acid into a well of a polypropylene microtiter plate having a plurality of wells;

adding reactants to said well, said reactants being for reaction of said nucleic acid in a gene amplification reaction, said reactants including at least one nucleic acid primer having a sequence selected so as to allow preferential amplification of said specific nucleic acid sequence;

conducting a gene amplification reaction on the nucleic acid in said well, thereby amplifying said specific nucleic acid sequence if present in said sample;

adding a dye to the well to form a conjugate with nucleic acid present in the well, said dye complexing with the nucleic acid sequence amplified and when optically excited producing a detectable emission in the visible spectrum, said dye having a DNA partition coefficient in a 10% ethanol/water solution of greater than 1×

10⁷ ; and

optically exciting the nucleic acid conjugated with the dye;

detecting and measuring the detectable emission from said dye without transferring the sample from said well in order to determine the quantity of the nucleic acid sequence that was amplified in each well;

determining a control level of detectable emission from a well in which nucleic acid has not been amplified; and

comparing the amount of detectable emission with the amount of detectable emission in said control, wherein an amount of detectable emission greater than in said control indicates the presence of the specific nucleic acid.

16. The method of claim 15, additionally comprising isolating the nucleic acid from the sample.

17. The method of claim 15, wherein the dye is selected from the group consisting of Yoyo-1, Toto-1 and Toto-3.

18. The method of claim 15, wherein the volume of the reactants and sample is between about 5 μ

l and about 50 μ

l.

19. The method of claim 15, wherein the gene amplification reaction is selected from the group consisting of polymerase chain reaction (PCR), self-sustained sequence replication (3SR), ligation amplification reaction system (LAR/LAS) and beta-Q replicase (β

QR).

20. The method of claim 15, wherein said nucleic acid sequence comprises a sequence unique to a particular organism.

21. The method of claim 20, wherein said organism is capable of causing infectious disease in a mammal.

22. The assay of claim 15, additionally comprising layering mineral oil over the reactants in the wells prior to conducting the gene amplification reaction.

23. The assay of claim 22, wherein said oil remains layered over the reactants during the detecting step.

26. The method of claim 15, wherein said dye is a thiazolium or azolium dye.