Device for identifying the presence of a nucleotide sequence in a DNA sample

• US 20020086309A1
• Filed: 08/24/2001
• Published: 07/04/2002
• Est. Priority Date: 04/29/1999
• Status: Abandoned Application

• Current Assignee: Genome Therapeutics Corporation
• Original Assignee: Genome Therapeutics Corporation
• Inventors: Manchec, Jean-Francois, Benn, James
• Application Number:
  US09/939,011
• Publication Number:
  US 20020086309A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/026 Fluid interfacing between devices or objects, e.g. connectors, inlet details
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2300/044 pierceable, e.g. films, membranes
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/50255 Multi-well filtration
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/50857 using arrays or bundles of open capillaries for holding samples
• CPC Class Codes: B01L 3/563 Joints or fittings in general F16L; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/125 : Allele specific primer extension
• CPC Class Codes: C12Q 2565/1015 : labels being on the same oligonucleotide

Abstract

A system and method for identifying the presence of specific nucleotide sequences in a target DNA sample is provided. The nucleotide detection system comprises a flat plate detection cell having a sample chamber, a membrane and an optical window providing optical access to the interior of the cell. A target DNA sample is mixed with labeled nucleotides and other chemistries, and undergoes a chemical reaction, such that if the DNA sample has the nucleotide, the resulting mixture will contain labeled nucleotides that have undergone a change in molecular weight. The reacted sample is applied to the sample chamber and the membrane in the flat plate nucleotide detection system is utilized to effect the separation of the smaller molecular weight labels from the sample. After separation, the presence of the label in either the sample chamber or the filtrate chamber, or on the membrane itself, is detected through the optical window to the sample chamber or through an optical window to the filtrate chamber to determine the presence or absence of the nucleotide sequence.

Claims

1. A flat plate nucleotide detection cell, comprising:

an upper flat plate;

a sample chamber formed along a bottom surface of said upper flat plate for holding a sample;

a membrane provided along a portion of said sample chamber for separating a sample in the sample chamber, and

an optical window provided in said upper flat plate, said optical window for permitting light to pass between the sample chamber and a detector for monitoring the sample chamber.

2. The flat plate nucleotide detection cell of claim 1, further comprising:

a syringe docking port in said upper flat plate fluidly coupled to the sample chamber.

3. The flat plate nucleotide detection cell of claim 2, said syringe docking port further comprising:

a seal for providing a fluid-tight seal after being pierced by a needle of a syringe;

a needle stop for preventing the needle from entering said sample chamber.

a needle guide formed in a funnel shape in said syringe docking port to guide the needle toward said sample chamber.

4. The flat plate nucleotide detection cell of claim 2, wherein said syringe docking port further comprises a seal capable of providing a fluid-tight seal after being pierced by a needle.

5. The flat plate nucleotide detection cell of claim 2, wherein said syringe docking port further comprises a needle stop capable of preventing a needle of a syringe from entering said sample chamber.

6. The flat plate nucleotide detection cell of claim 2, further comprising a needle guide formed in said syringe docking port to guide a needle of a syringe toward said sample chamber.

7. The flat plate nucleotide detection cell of claim 6, wherein said needle guide is funnel shaped.

8. The flat plate nucleotide detection cell of claim 1, further comprising a vent hole in fluid communication with the sample chamber providing a vent for the sample chamber.

9. The flat plate nucleotide detection cell of claim 1, wherein the membrane comprises a flat sheet.

10. The flat plate nucleotide detection cell of claim 1, further comprising a filtrate chamber mated to said sample chamber via said membrane.

11. The flat plate nucleotide detection cell of claim 1, further comprising a lower flat plate coupled to the upper flat plate and forming a filtrate chamber, wherein the membrane is mounted between the lower flat plate and the upper flat plate, such that the sample chamber and the filtrate chamber are separated by the membrane.

12. The flat plate nucleotide detection cell of claim 11, wherein the lower flat plate includes a second optical window capable of transmitting light between the filtrate chamber and a detector for monitoring the filtrate chamber.

13. The flat plate nucleotide detection cell of claim 12, wherein the filtrate chamber is offset from the sample chamber.

14. The flat plate nucleotide detection cell of claim 1, wherein the sample chamber is serpentine.

15. The flat plate nucleotide detection cell of claim 14, wherein the sample chamber is S-shaped.

16. The flat plate nucleotide detection cell of claim 1, wherein the membrane has a molecular cut-off such that a labeled nucleotide excision product passes through the membrane.

18. The flat plate nucleotide detection cell of claim 17, further comprising:

a syringe docking port in said upper flat plate fluidly coupled to the sample chamber.

19. The flat plate nucleotide detection cell of claim 18, wherein said syringe docking port further comprises:

a seal for providing a fluid-tight seal after being pierced by a needle of a syringe;

a needle stop for preventing the needle of the syringe from entering said sample chamber.

a needle guide formed in a funnel shape in said syringe docking port to guide the needle of the syringe toward said sample chamber.

20. The flat plate nucleotide detection cell of claim 18, wherein said syringe docking port further comprises a seal for providing a fluid-tight seal after being pierced by a needle.

21. The flat plate nucleotide detection cell of claim 18, wherein said syringe docking port further comprises a needle stop for preventing a needle from entering said sample chamber.

22. The flat plate nucleotide detection cell of claim 18, further comprising a needle guide formed in said syringe docking port to guide a needle toward said sample chamber.

23. The flat plate nucleotide detection cell of claim 22, wherein said needle guide is funnel shaped.

24. The flat plate nucleotide detection cell of claim 17, further comprising a vent hole in fluid communication with the sample chamber providing a vent for the sample chamber.

25. The flat plate nucleotide detection cell of claim 17, wherein the membrane comprises a flat sheet.

26. The flat plate nucleotide detection cell of claim 17, wherein the filtrate chamber is offset from the sample chamber.

27. The flat plate nucleotide detection cell of claim 17, wherein the sample chamber is serpentine.

28. The flat plate nucleotide detection cell of claim 27, wherein the sample chamber is S-shaped.

30. The system of claim 29, wherein the interior chamber comprises a sample chamber holding a biological sample, and the optical detector monitors the biological sample.

31. The system of claim 29, wherein the interior chamber comprises a filtrate chamber for collecting a filtrate from the membrane, and the optical detector monitors the filtrate.

17. A flat plate nucleotide detection cell, comprising

an upper flat plate;

at least one sample chamber formed along a bottom surface of said upper flat plate;

a lower flat plate forming a filtrate chamber,

a membrane for separating a sample provided along a portion of said sample chamber and mounted between the upper channel plate and the lower channel plate such that the sample chamber and the filtrate chamber at least partially overlap; and

an optical window provided in said lower channel plate, said optical window for permitting light to pass between the filtrate chamber and a detector for monitoring the filtrate chamber.

29. A system for detecting the presence of a nucleotide sequence in a DNA sample, comprising:

a flat plate detection cell having an interior chamber, a membrane provided along a portion of the interior chamber and an optical window providing access to the interior chamber; and

an optical detector positioned relative to the optical window to monitor the interior chamber.

32. A method of detecting the presence of a nucleotide sequence in a DNA sample, comprising,

providing a flat plate detection cell having a sample chamber, a filtrate chamber, a membrane provided between the sample chamber and the filtrate chamber and having a molecular weight cut-off such that a labeled nucleotide excision product passes through the membrane and an optical window providing access to one of the sample chamber and the filtrate chamber;

injecting an admixture into said sample chamber and into contact with a first side of said membrane, said admixture comprising a hybridization product formed of a primer and a strand of DNA in said sample, wherein the primer comprises a sequence of DNA which hybridizes with said strand of DNA adjacent to said first nucleotide position and having a second nucleotide opposite said first nucleotide position, said second nucleotide associated with a label, said second nucleotide hybridizing to said first nucleotide in the event said second nucleotide is complementary to said first nucleotide and said second nucleotide not hybridizing to said first nucleotide in the event said second nucleotide is not complementary, and wherein a proofreading polymerase has been applied to the hybridization product under conditions in which said second nucleotide is preferentially excised to form a labeled nucleotide excision product in the event said second nucleotide is not hybridized to said first nucleotide, and in which said second nucleotide is preferentially incorporated into an extension product in the event said second nucleotide is hybridized to said first nucleotide;

applying one of a dialysis solution to the second side of the membrane and a pressure differential to the sample chamber along the first side of the membrane to pass a labeled nucleotide excision product through the membrane; and

monitoring at least one of the group of;

the sample on the first side of the ultrafiltration membrane and a filtrate on the second side of the ultrafiltration membrane, for the presence of a label through said optical window, wherein the presence of a label in the filtrate in concentrations greater than a background amount after a first predetermined time period is indicative of the absence of the first nucleotide, and the presence of a label remaining in the ultrafiltration chamber in concentrations greater than a background amount after a second predetermined time period greater than said first predetermined time period is indicative of the presence of the first nucleotide.