Purification method and apparatus

• US 6,919,200 B2
• Filed: 11/22/1999
• Issued: 07/19/2005
• Est. Priority Date: 11/23/1998
• Status: Expired due to Fees

• Current Assignee: NAArmy, U.S. As Represented By The Secretary, The
• Original Assignee: The United States Of America As Represented By The Secretary Of The Army
• Inventors: Ibrahim, Sofi M.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US09/444,095
• Publication Number:
  US 20010036630A1
• Time in Patent Office: 2,066 Days
• Field of Search: 435/6, 435/270, 435/287.2, 435/287.3, 435/91.1, 435/91.2, 435/270.1, 436/94, 436/501, 536/23.1, 422/57, 422/58, 422/99, 530/587.2
• US Class Current: 435/270
• CPC Class Codes: B01L 3/508 rigid containers not provided for above
• 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 2001/028 Sampling from a surface, swabbing, vaporising
• CPC Class Codes: G01N 2001/2826 Collecting by adsorption or absorption
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A purification apparatus, kit and method for purifying DNA, RNA, proteins, antigens, antibodies and cells. The apparatus has a wand and a reservoir tube. The wand is made of a cap, a sample collection assembly and an elongated shaft connecting the cap to the sample collection assembly. The sample collection assembly has a series of microstructures on its surface, or microparticles enclosed within it for increasing the surface area of the sample collection assembly. The increased surface area permits maximum exposure to and binding of target molecules thereto. The reservoir tube associated with the wand has one end defining an opening and a second end that is closed and preferably cone or cylindrical shaped. The cap of the wand securely and sealingly fastens to the open end of the reservoir tube with the shaft and the sample collection assembly fitting easily inside the reservoir tube. The apparatus, kit and methods can be used for protein and nucleic acids detection by calorimetric, luminescent, fluorescent or electrochemical means through attachments for detecting such signals. The apparatus, kit and methods can also be used in conjunction with an attachment for thermal regulation to perform nucleic acids amplification. The apparatus, kit and methods can further be configured for integrated, high throughput purification and detection of proteins and nucleic acids.

Claims

1. A method of DNA or RNA purification comprising:

placing a DNA or RNA containing sample in a first reservoir tube with a solution to effect release of DNA or RNA from cells in said sample;

inserting a wand into said first reservoir tube, wherein said wand comprises a cap, a sample collection assembly and an elongated shaft connecting said cap to said sample collection assembly, said sample collection assembly having microstructures for increasing the surface area of the sample collection assembly;

securely and sealingly closing said first reservoir tube with said cap of said wand with said shaft and said sample collection assembly inside said first reservoir tube;

agitating said first reservoir tube to mix said sample with said solution under conditions for releasing said DNA or RNA front cells in said sample and non-specifically binding said DNA or RNA to said microstructures of said sample collection assembly, thereby non-specifically binding said DNA or said RNA to said microstructures of said sample collection assembly;

removing said wand from said first reservoir tube and inserting said wand into a second reservoir tube, said second reservoir tube containing a wash buffer;

securely and sealingly closing said second reservoir tube with said cap of said wand with said shaft and said sample collection assembly inside said second reservoir tube;

agitating said second reservoir tube to mix said sample with said wash buffer under conditions to retain only said DNA or said RNA on said microstructures;

removing said wand front said second reservoir tube and inserting said wand into a third reservoir tube, said third reservoir tube containing an elution buffer, wherein said elution buffer causes release of said nucleic acids from said microstructures;

incubating said third reservoir tube; and

recovering purified DNA or RNA from said third reservoir tube.

2. The method of claim 1, wherein said sample capture assembly comprises a main body having one or more flanges with microstructures for binding target molecules.

3. The method of claim 2, wherein said microstructures are selected from the group consisting of cross-etched lanes, dimples, pillars and pores.

4. The method of claim 1, wherein said microstructures are selected from the group consisting of cross-etched lanes, dimples, pillars and pores.

5. The method of claim 1, wherein said sample collection assembly comprises a mesh outer surface wherein said microstructures are microparticles enclosed within said mesh outer surface.

6. The method of claim 1, wherein said microstructures of said sample collection assembly are coated with a material that binds non-specifically with nucleic acids.

7. The method of claim 6, wherein said material is silicon oxide or aluminum oxide.

8. The method of claim 1, wherein said microstructures comprise deep reactive ion etchings or toolings that provide an increased surface area on said sample collection assembly.

9. A method of purifying specific DNA or RNA comprising:

placing a purified DNA or RNA sample in a first reservoir tube under conditions to denature double stranded DNA or render RNA suitable for binding;

inserting a wand into said first reservoir tube, wherein said wand comprises a cap, a sample collection assembly and an elongated shaft connecting said cap to said sample collection assembly, said sample collection assembly having microstructures for increasing the surface area of the sample collection assembly, and said microstructures of said sample collection assembly are coated with a coating comprising sequence specific oligonucleotide probe, peptide nucleic acid probe through a linker arm, or biotin-streptavidin bond to capture specific target DNA or RNA;

securely and sealingly closing said first reservoir tube with said cap of said wand with said shaft and said sample collection assembly inside said first reservoir tube, and incubating said DNA or said RNA of the sample in the sample collection assembly under conditions whereby stable, specific hybridization structures are formed, thereby binding said specific DNA or said specific RNA to said coating on said microstructures of said sample collection assembly;

removing said wand from said first reservoir tube and inserting said wand into a second reservoir tube, said second reservoir tube containing a wash buffer;

securely and sealingly closing said second reservoir tube with said cap of said wand with said shaft and said sample collection assembly inside said second reservoir tube;

agitating said second reservoir tube to mix said sample with said wash buffer under conditions to retain only said DNA or said RNA on said microstructures;

removing said wand from said second reservoir tube and inserting said wand into a third reservoir tubes said third reservoir tube containing an elution buffer to effect release of said DNA or said RNA;

incubating said third reservoir tube;

removing said sample collection assembly from said third reservoir tube;

recovering said specific DNA or RNA from said third reservoir tube.

10. The method of claim 9, wherein said conditions for denaturing DNA or rendering RNA suitable for binding comprise:

heating said reservoir tube for a sufficient time to denature said double stranded DNA or render said RNA suitable for binding.

11. The method of claim 9, wherein said microstructures comprise deep reactive ion etchings or toolings that provide an increased surface area on said sample collection assembly.

12. The method of claim 9, wherein said DNA coating is single stranded DNA and double stranded hybridization structures are formed.

13. The method of claim 9, wherein said DNA coating is double stranded DNA and triplex hybridization structures are formed.

14. A method of purifying specific DNA or RNA comprising:

placing a purified DNA or RNA sample in a first reservoir tube under conditions to denature double stranded DNA or render RNA suitable for binding;

inserting a wand into said first reservoir tube, wherein said wand comprises a cap, a sample collection assembly and an elongated shaft connecting said cap to said sample collection assembly, said sample collection assembly having microstructures for increasing the surface area of the sample collection assembly, and said microstructures of said sample collection assembly are coated with a coating comprising sequence specific oligonucleotide probe, peptide nucleic acid probe through a linker arm, or biotin-streptavidin bond to capture specific target DNA or RNA;

securely and sealingly closing said first reservoir tube with said cap of said wand with said shaft and said sample collection assembly inside said first reservoir tube, and incubating said DNA or said RNA of the sample in the sample collection assembly under conditions whereby stable, specific hybridization structures are formed, thereby binding said specific DNA or said specific RNA to said coating on said microstructures of said sample collection assembly;

removing said wand from said first reservoir tube and inserting said wand into a second reservoir tube, said second reservoir tube containing a wash buffer;

securely and sealingly closing said second reservoir tube with said cap of said wand with said shaft and said sample collection assembly inside said second reservoir tube;

agitating said second reservoir tube to mix said sample with said wash buffer under conditions to retain only said DNA or said RNA on said microstructures;

removing said wand from said second reservoir tube and inserting said wand into a third reservoir tube;

heating said third reservoir tube under conditions to effect release of said DNA or said RNA from said microstructures;

removing said sample collection assembly from, said third reservoir tube; and

recovering said specific DNA or RNA from said third reservoir tube.