Eucaryotic genomic dna dot-blot hybridization method

Claim 1

A method for detecting a particular base sequence in a sample of eucaryotic genomic DNA by nucleic acid hybridization, comprising the steps of: (a) immobilizing DNA from the sample in single stranded form in a limited zone of a solid support sheet, (b) treating the support sheet to substantially block unoccupied nucleic acid binding sites thereon, (c) incubating the support with a solution containing an oligonucleotide probe under hybridizing conditions, such probe having a base sequence exactly complementary to the base sequence of interest in the sample, (d) separating the excess probe solution from the support, (e) washing the support at a stringency sufficient to remove substantially all probe which had hybridized to sequences other than the exactly complementary sequence of interest, and (f) detecting the presence of hybridized probe in said zone of the support sheet.

Claim 2

A method for detecting a particular base sequence in a sample of eucaryotic genomic DNA by nucleic acid hybridization, comprising the steps of: (a) immobilizing DNA from the sample in single stranded form in a limited zone of a solid support sheet, (b) treating the support sheet to substantially block unoccupied nucleic acid binding sites thereon, (c) incubating the support with a solution containing an oligonucleotide probe under hybridizing conditions, such probe having a base sequence exactly complementary to the base sequence of interest in the sample, (d) separating the excess probe solution from the support, (e) washing the support at a stringency sufficient to remove substantially all probe which had hybridized to sequences other than the exactly complementary sequence of interest, and (f) detecting the presence of hybridized probe in said zone of the support sheet.

Claim 4

The method of any of claims 1 and 2 wherein the concentration of the probe in the solution incubated with the support to which the single stranded sample DNA is immobilized is between about 10 pM and 10 nM.

Claim 5

The method of any of claims 1 to 3 wherein the sample is a liquid mixture, is treated to denature DNA therein and applied to the solid support sheet, and residual liquid removed by vacuum or the sample is applied to the solid support sheet and DNA therein is denatured in situ.

Claim 6

The method of any of claims 1 to 4 wherein the probe comprises a detectable label which is detected in said zone of the support sheet in order to detect the presence of hybridized probe.

Claim 7

A method for detecting a particular normal or mutated gene in a human patient by a dot-blot hybridization, comprising the steps of: (a) obtaining a sample from the patient which contains nucleated cells, (b) isolating essentially unfractionated genomic DNA from cells contained in such sample, (c) denaturing DNA in the unfractionated genomic DNA isolate by treatment with base to provide genomic DNA in single stranded form, (d) neutralizing the base-treated liquid isolate, (e) applying the neutralized liquid isolate to a solid support sheet in two separate limited zones, the support sheet being capable of immobilizing single stranded DNA, and removing residual liquid by application of a vacuum to the reverse side of the support sheet, (f) contacting the support sheet for a predetermined time with a prehybridization solution comprising agents which bind to and block unoccupied nucleic acid binding sites on the sheet, (g) cutting the support sheet into two sections; one comprising the first zone and the other comprising the second zone, (h) contacting one of the cut sections of the support sheet with a hybridization solution comprising an oligonucleotide probe having a base sequence exactly complementary to the normal sequence of the gene of interest and contacting the other section of the support sheet with a hybridization solution comprising an oligonucleotide probe having a base sequence exactly complementary to the mutated sequence, each of the probes comprising a detectable label, (i) incubating the respective support sections in contact with the hybridization solutions for less than about four hours at a temperature favorable to hybridization between the probes and their complementary sequences, (j) removing the hybridization solutions from the sections of the support sheet, (k) washing the support sheet sections at a stringency sufficient to remove substantially all probe which had hybridized to sequences other than their exactly complementary sequences, (1) detecting the presence of the probe label in said first and second zones of the cut support sheet sections.

Claim 8

A method for detecting a particular normal or mutated gene in a human patient by a dot-blot hybridization, comprising the steps of: (a) obtaining a sample from the patient which contains nucleated cells, (b) isolating essentially unfractionated genomic DNA from cells contained in such sample, (c) denaturing DNA in the unfractionated genomic DNA isolate by treatment with base to provide genomic DNA in single stranded form, (d) neutralizing the base-treated liquid isolate, (e) applying the neutralized liquid isolate to a solid support sheet in two separate limited zones, the support sheet being capable of immobilizing single stranded DNA, and removing residual liquid by application of a vacuum to the reverse side of the support sheet, (f) contacting the support sheet for a predetermined time with a prehybridization solution comprising agents which bind to and block unoccupied nucleic acid binding sites on the sheet, (g) cutting the support sheet into two sections; one comprising the first zone and the other comprising the second zone, (h) contacting one of the cut sections of the support sheet with a hybridization solution comprising an oligonucleotide probe having a base sequence exactly complementary to the normal sequence of the gene of interest and contacting the other section of the support sheet with a hybridization solution comprising an oligonucleotide probe having a base sequence exactly complementary to the mutated sequence, each of the probes comprising a detectable label, (i) incubating the respective support sections in contact with the hybridization solutions for less than about four hours at a temperature favorable to hybridization between the probes and their complementary sequences, (j) removing the hybridization solutions from the sections of the support sheet, (k) washing the support sheet sections at a stringency sufficient to remove substantially all probe which had hybridized to sequences other than their exactly complementary sequences, (1) detecting the presence of the probe label in said first and second zones of the cut support sheet sections.