Methods of extracting, amplifying and detecting a nucleic acid from whole blood or PBMC fraction

• US 5,334,499 A
• Filed: 09/12/1989
• Issued: 08/02/1994
• Est. Priority Date: 04/17/1989
• Status: Expired due to Term

• Current Assignee: Clinical Diagnostic Systems Inc.
• Original Assignee: Eastman Kodak Company
• Inventors: Burdick, Brent A., Ekeze, Tobias D.
• Primary Examiner(s): YARBROUGH, AMELIA
• Application Number:
  US07/406,222
• Time in Patent Office: 1,785 Days
• Field of Search: 435/6, 435/91, 435/501, 435/803, 935/77, 935/78, 935/6, 424/520, 436/177
• US Class Current: 435/5
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 2740/16022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

A nucleic acid is rapidly extracted from whole blood or a peripheral blood mononuclear cell (PBMC) fraction thereof. Extraction from the PBMC fraction is accomplished by heating the fraction at or near the boiling point of water for a few minutes and recovering the extracted nucleic acid. This rapid method is particularly useful for extracting DNA for the detection of genetic diseases or infectious agents, such as HIV-I. Whole blood can likewise be heated after it is mixed with a salt solution containing a polysaccharide, such as dextran. The extracted nucleic acid is then recovered from the heated mixture. Nucleic acids extracted in this way are available for amplification using a polymerase chain reaction. Where the presence of a specific gene is to be determined for diagnostic purposes, it can be extracted as described above and subjected to suitable amplification and detection steps.

Claims

1. A method for the rapid extraction of a nucleic acid from a specimen of whole blood, said method consisting of:

A. mixing a whole blood specimen with a salt solution comprising one or more polysaccharides,

B. subjecting the mixture resulting from step A to heat at a temperature at least at or near the boiling point of water for from about two to about fifteen minutes to lyse the cells in said mixture to release a nucleic acid from said cells, and

C. recovering said nucleic acid from said heated mixture.

2. The method of claim 1 wherein said polysaccharide is a dextran present in an amount of from about 1 to about 10 weight percent of said whole blood specimen.

3. The method of claim 1 wherein said salt solution further comprises a nonionic surfactant.

4. The method of claim 1 for the extraction of either Human β

-globin or Human Leukocyte Antigen DNA.

5. The method of claim 1 wherein said mixture is heated for from about four to about twelve minutes at a temperature of from about 95°

to about 120°

C.

6. The method of claim 2 wherein said nucleic acid is extracted from a dried whole blood specimen.

7. The method of claim 1 for the extraction of a nucleic acid from cells which have been infected by an agent selected from the group consisting of herpes, hepatitis, rubella, Epstein Barr virus, cytomegalovirus and a retrovirus.