Detection of particular nucleotide sequences

• US 4,767,700 A
• Filed: 02/15/1985
• Issued: 08/30/1988
• Est. Priority Date: 02/15/1985
• Status: Expired due to Term

• Current Assignee: Beckman Research Institute of the City of Hope (City of Hope)
• Original Assignee: Beckman Research Institute of the City of Hope (City of Hope)
• Inventors: Wallace, R. Bruce
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Jay, Jeremy M.
• Application Number:
  US06/702,099
• Time in Patent Office: 1,292 Days
• Field of Search: 435/6, 435/803, 935/78, 436/501, 436/811
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

A process for the detection of a particular polynucleotide sequence in a polynucleotide-containing test sample comprising forming an agarose gel having a depression therein, adding the test sample to said depression, putting agarose solution in the depression and permitting it to gel thereby to encase the test sample in agarose gel, denaturing the polynucleotide in the test sample within the agarose gel, under hybridization conditions contacting the test sample-containing gel with an oligonucleotide or polynucleotide probe having a sequence complementary with a sequence for which the sample is being tested, and determining the extent of hybridization.

Claims

1. A process for the detection of a particular polynucleotide sequence in a polynucleotide-containing test sample consisting essentially of forming an agarose gel having a depression therein, adding the test sample to said depression, putting agarose solution in the depression and permitting it to gel thereby encasing the test sample in agarose gel, denturing the polynucleotide in the test sample within the agarose gel, contacting the test sample-containing gel with an oligonucleotide or polynucleotide probe having a sequence complementary with a sequence for which the sample is being tested under hybridization conditions, said probe having at least 23 nucleotides, said probe carrying a readable label, and determining the extent of hybridization, the extent of hybridization being determined by reading the label and, wherein after hybridization the gel is washed to remove non-hybridized probe.

2. A proces according to claim 1, wherein the hybridization is conducted in 5×

SSPE, wherein SSPE comprises 0.18M NaCl, 10 mM, (Na₁.5) PO₄, 1 mM Na₂ EDTA, pH of 7.0, 0.1% SDS and 10 μ

g/m;

of sonicated, denatured E. coli DNA for 3 hours at 60°

C.

3. A process according to claim 1 wherein the label is a radioactive isotope.

4. A process according to claim 1 wherein the label is biotin.

5. A process according to claim 1, wherein the encased test sample is dried prior to denaturing.

6. A process according to claim 1, wherein the probe is an oligonucleotide.

7. A process according to claim 1, wherein the probe is a polynucleotide sequence which distinguishes between different sequences in test samples from individuals suspected of having a particular genetic condition.

8. A process according to claim 7, wherein the genetic condition is sickle cell anemia.

9. A process according to claim 7, wherein the genetic condition is thalassemia.