Containment cuvette for PCR and method of use

• US 5,229,297 A
• Filed: 10/15/1992
• Issued: 07/20/1993
• Est. Priority Date: 02/03/1989
• Status: Expired due to Fees

• Current Assignee: Clinical Diagnostic Systems Inc.
• Original Assignee: Eastman Kodak Company
• Inventors: Schnipelsky, Paul N., Hinckley, Charles C., Wellman, Jeffrey A., Seaberg, Leonard J., Findlay, John B., Donish, William H.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Le, Long V.
• Application Number:
  US07/962,159
• Time in Patent Office: 278 Days
• Field of Search: 435/6, 435/91, 435/172.3, 435/301, 436/63, 436/86, 436/94, 436/180, 436/501, 436/508, 436/808, 436/809, 436/815, 935/77, 935/78
• US Class Current: 436/94
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A cuvette and a method of use which prevent nucleic acid amplified by PCR technology from being released to the atmosphere, while still proceeding to a detection step to determine whether or not the nucleic acid is present. Detection reagents are either pre-incorporated into compartments in the cuvette or added after amplification. In the latter case, a check valve prevents amplified nucleic acid from being released. Transfer of liquids between compartments is achieved via the use of flexible compartment walls and an external pressure source, or via pistons that are part of the cuvette and operate on the compartments as a piston within a piston chamber.

Claims

1. A method for amplifying and detecting nucleic acid material in a closed cuvette without allowing aerosols to exit therefrom to contaminate the environment, the method comprising the steps of

a) providing within a reaction compartment of a cuvette a sample of nucleic acid material and amplifying reagents, said cuvette comprising a plurality of compartments including said reaction compartment and storage means for storing a detection material, at least one of said compartments including a detection site, and means for interconnecting said compartments to provide fluid transfer;

b) closing off permanently the portions of said cuvette containing the nucleic acid material to lock all nucleic acid into said cuvette;

c) amplifying the nucleic acid material by cycling said reaction compartment through temperature changes preselected to cause said reagents to be effective to amplify said temperature changes including temperatures exceeding 37°

C.;

d) fluidly transferring amplified nucleic acid material and detection material to said detection site while keeping said cuvette closed against leakage of nucleic acid material outside of the cuvette; and

e) detecting the amplified nucleic acid material at said detection site with said detection material, all while the nucleic acid material remains confined within said cuvette.

3. A method as defined in claim 1 or 2, wherein said step c) comprises the step of transferring heat across a wall of said reaction compartment, both into and out of said compartment, said wall comprising at least one thermally conductive material.

4. A method as defined in claim 3, wherein said wall has a thermal path length of no more than about 0.3 mm and a thermal resistance of no more than about 5.0°

C./watt.

5. A method as defined in claim 1 or 2, wherein at least one wall of said compartments is sufficiently flexible as to allow external pressure to compress said compartments to force liquid transfer out of said compartments, and wherein said step d) comprises the step of applying exterior pressure to said flexible walls of said compartments in a predetermined sequence.

6. A method as defined in claim 1 or 2, wherein said detection material include a bead comprising a magnetizable material and wherein said steps d)-e) comprise the steps of transferring said beads to said reaction compartment, attaching said detection material, including said beads, to said amplified nucleic acid material and washing away unattached detection material in the presence of a magnetic field that retains said beads and attached detection material within said reaction compartment.

7. A method as defined in claim 1, wherein said steps d) and e) occur sequentially by pressurizing first said reaction compartment and thereafter a storage compartment.

8. A method as defined in claim 1, wherein said steps d) and e) occur by pressurizing said storage compartment and said reaction compartment simultaneously, and retarding the flow of detection material until amplified nucleic acid material has been transferred to said site.

9. A method as defined in claim 1, and further including as a step prior to said step e), the step of reconstituting detection material deposited in dried form in a storage compartment, by transferring pre-incorporated water to said dried material from a storage compartment.

10. A method as defined in claim 1, wherein step a) comprises the step of injecting at least blood cells and optional DNA extraction agents into a predetermined one of said compartments to form a solution;

and before step c), further including the steps of;

i) extracting DNA from the cells in said predetermined compartment; and

ii) after a suitable incubation period, forcing said solution of extracted DNA and cell fragments through a filter disposed between said predetermined one compartment and said reaction compartment, said filter being sized to retain cellular fragments and to pass DNA.

11. A method as defined in claim 1 or 2, wherein said amplifying reagents include a polymerase and said amplifying step includes extending a primer annealed to a DNA strand by the action of said polymerase, to form a double-stranded DNA.

12. A method as defined in claim 11, wherein said extending step is followed by heating at a predetermined temperature to separate the newly formed double-stranded DNA into single strands.

13. A method as defined in claim 1, wherein said cuvette further includes a first piston chamber and a first piston in said chamber, fluidly connected to said reaction compartment so that the advance of said piston in said chamber causes pressure to be increased in said reaction compartment, and a second piston chamber and piston therein, fluidly connected to said detection site so that when said second piston is withdrawn in its chamber, it relieves pressure at said detection site and said step d) comprises the step of advancing said first piston while withdrawing said second piston.

14. A method as defined in claims 1 or 13 wherein said step of detecting is done while heating said detection site in an amount sufficient to hybridize reagents used for said detecting.

15. A method as defined in claim 13, wherein said detection reagents are incorporated into the cuvette prior to said step c).

2. A method for amplifying and detecting nucleic acid material in a closed cuvette without allowing aerosols to exit therefrom to contaminate the environment, the method comprising the steps of

a) placing a sample suspected of containing a target nucleic acid material, and amplifying reagents, into a cuvette comprising a reaction compartment, a detection site, reagents effective to provide detection of the target nucleic acid material, and means allowing transfer of amplified nucleic acid material to said detection site;

b) closing off permanently the portions of said cuvette containing the nucleic acid material to lock all nucleic acid material into said cuvette;

c) amplifying the nucleic acid material by cycling said reaction compartment through temperature changes that include temperatures in excess of 37°

C. and which are preselected to cause said reagents to be effective;

d) fluidly transferring amplified nucleic acid material to said detection site;

e) interacting at said detection site, any amplified target nucleic acid material with detection reagents;

f) detecting the amplified nucleic acid material at such detection site, and

g) during steps c) through f), maintaining the cuvette closed to the atmosphere so that all nucleic acid material remains confined within the cuvette and carry-over contamination is prevented.