SYSTEM FOR ISOLATING BIOMOLECULES FROM A SAMPLE

• US 20080153078A1
• Filed: 12/19/2007
• Published: 06/26/2008
• Est. Priority Date: 06/15/2006
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Chang, James, BRAMAN, Jeffrey C., Basehore, Lee Scott, Novoradovskaya, Natalia
• Application Number:
  US11/959,587
• Publication Number:
  US 20080153078A1
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: B01L 2200/026 Fluid interfacing between devices or objects, e.g. connectors, inlet details
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0644 rotary valves
• 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: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 1/405 by adsorption or absorption

Abstract

The present invention provides an automated system for purification of a substance of interest. The system generally comprises an instrument for moving fluids through the system, a reagent pack for storing fluids, and a purification cartridge. The cartridge comprises two filtration units for binding substances based on different physical properties. The cartridge also comprises rotary valves for control of movement of fluids on the cartridge. In preferred embodiments, the system is useful for purifying RNA from blood samples.

Claims

1. An article of manufacture for purification of RNA from white blood cells of a sample comprising white blood cells, said article comprising:

at least one port for intake of each of a number of fluids;

at least one port for exit of at least one fluid;

at least one solid support for binding of cells of whole blood;

at least one solid support for binding of RNA from cells of whole blood; and

at least one rotary valve for control of movement of fluids among three or more conduits,

wherein the intake port(s), exit port(s), solid supports, and rotary valve(s) are connected to each other to create a circuit from the intake port to the exit port.

2. The article of claim 1, wherein the sample comprises whole blood of an animal.

3. The article of claim 2, wherein the animal is a human.

4. The article of claim 1, which comprises ten intake ports and one exit port.

5. The article of claim 1, which comprises four rotary valves that function independently of each other.

6. The article of claim 1, wherein the rotary valve(s) are computer controllable.

7. The article of claim 1, wherein the solid support for binding of RNA is a glass fiber filter.

21. A system for purification of RNA from white blood cells of a sample comprising white blood cells, said system comprising:

the article of manufacture of claim 1;

at least one pump for movement of fluids into, through, and out of the article of manufacture;

a reagent pack for storing fluids to be moved through the article of manufacture; and

a computer for controlling movement of fluids through the article of manufacture.

22. The system of claim 21, wherein the pump comprises more than one head and is capable of creating both a positive pressure on a fluid and a negative pressure on a fluid.

23. The system of claim 21, wherein the pump is a peristaltic pump.

24. The system of claim 21, wherein the reagent pack comprises two or more containers for containing two or more different fluids, each of which containers is capable of being connected to a port in the article of manufacture by way of a connector.

25. The system of claim 21, wherein the computer controls the movement of the pump(s) and controls movement of valves on the article of manufacture.

26. The system of claim 21, further comprising a waste pack for receiving and containing waste from the article of manufacture.

8. An automated method for the isolation of RNA from white blood cells, said method comprising:

a) causing a sample comprising white blood cells to contact and flow over a first solid support, whereby the first solid support entraps cells present in the sample and removes them from the sample;

b) causing cells other than white blood cells on the first solid support to lyse, whereby lysis causes the cells and their components to be released from the first solid support and removed from cells remaining entrapped by the first solid support;

c) causing the cells remaining on the first solid support to lyse, thereby releasing RNA into a lysate; and

d) causing the lysate to contact and flow over a second solid support, whereby the second solid support binds the RNA and allows other substances to pass unbound,

wherein the method is performed on a single device and the movement of fluids within the method is controlled, at least in part, by two or more rotary valves present on the device, and

wherein all of the steps of the method are controlled automatically by a computing means.

9. The method of claim 8, further comprising:

causing the bound RNA to elute from the second solid substrate; and

collecting the eluted RNA.

10. The method of claim 9, wherein the method comprises:

between the steps of binding of RNA to the second solid substrate and causing the bound RNA to elute from the second solid substrate, exposing the bound RNA to a liquid comprising ethanol,

wherein the method does not include exposing the bound RNA to an aqueous composition between exposing it to an ethanol-containing liquid and eluting it from the second solid substrate.

11. The method of claim 10, wherein RNA bound to the second solid substrate is treated with ethanol then eluted with water or a low ionic strength aqueous solution, without an intervening wash with an aqueous composition.

12. The method of claim 8, wherein the sample comprises whole blood.

13. The method of claim 8, wherein the white blood cells are cultured or transformed white blood cells.

14. The method of claim 8, wherein the method is performed in 15 minutes or less.

15. The method of claim 8, wherein movement of fluids is automatically controlled by the computing means at least in part by regulating positive pressure to push the fluids and negative pressure to pull fluids.

16. The method of claim 8, wherein movement of fluids is automatically controlled by the computing means at least in part by regulating the positioning of the rotary valves to allow selective opening and closing of conduits for movement of fluids through the device.

17. The method of claim 8, wherein the device comprises four rotary valves.

18. The method of claim 8, wherein the method comprises:

mixing whole blood with a composition that causes lysis of red blood cells;

exposing the mixture to a first solid support that entraps blood cells;

exposing the entrapped blood cells to a composition that causes lysis of red blood cells to effect lysis of a substantial amount of red blood cells entrapped on the first solid support;

washing the first solid support at least one time with an aqueous solution to remove entrapped cell debris and blood components other than white blood cells;

optionally, passing a gas over the first solid substrate to effect partial or total drying of the substrate;

exposing the entrapped white blood cells to a composition that causes the white blood cells to lyse;

collecting the lysate;

exposing the first solid substrate to water to elute additional cell lysis substances;

collecting the water-containing composition;

mixing the lysate and water-containing composition;

combining the lysate-water mixture with sulfolane to make an RNA binding solution;

exposing the RNA binding solution to a second solid substrate, which binds the RNA in the solution;

optionally exposing the bound RNA to an aqueous solution to wash off impurities;

exposing the bound RNA to ethanol.

19. The method of claim 18, further comprising storing the bound RNA in ethanol for at least one day.

20. The method of claim 18, further comprising exposing the bound RNA to water or an aqueous solution to elute the RNA from the second solid substrate.