US 4,683,194 A · Method for detection of polymorphic restriction sites and nucleic acid sequences

Abstract

In a method for detecting the presence or absence of a specific restriction site in a nucleic acid sequence an oligonucleotide probe complementary to one strand of the nucleic acid sequence spanning said restriction site is synthesized. The probe is labeled at the end nearer the restriction site. The nucleic acid is hybridized to the probe and a blocking oligomer may be added, if necessary, to prevent non-specific binding of the probe. Subsequent digestion with a restriction enzyme cleaves those oligomers that have hybridized to the nucleic acid and reformed the restriction site. The resulting cut and uncut labeled oligomers are separated and detected based on the type of probe label. The described method may be used to detect sickle cell anemia.

Claims28

Dependent claims

A method for detecting the presence or absence of at least one specific restriction site in a specific nucleic acid sequence comprising the steps of:

(a) hybridizing said nucleic acid sequence in solution with an oligonucleotide probe for each restriction site being detected which probe is complementary to a region in said nucleic acid sequence spanning the respective restriction site of the probe and which probe is labeled at the end which is nearer to said respective restriction site than the other end of the probe;
(b) digesting said hybridized nucleic acid sequence with a restriction endonuclease for each restriction site being detected by each probe which is capable of cleaving its respective probe at said restriction site being detected so as to produce labeled and unlabeled oligomer fragments;
(c) separating any labeled cleaved oligomer fragments from labeled uncleaved oligomers;
and
(d) detecting the presence or absence of labeled oligomer fragments.

2 more elements

A method for detecting the presence or absence of one or both alleles of a polymorphic restriction site in a specific nucleic acid sequence comprising the steps of:

(a) hybridizing said nucleic acid sequence in solution with an oligonucleotide probe which is complementary to a region in said nucleic acid sequence spanning a first restriction site present in only one allele and a second restriction site commonto both alleles, and which probe is labeled at the end which is nearer to one of said restriction sites than the other end of the probe;
(b) digesting said hybridized nucleic acid sequence with a first restriction endonuclease which is capable of cleaving said probe only at said first restriction site so as to produce labeled and unlabeled oligomer fragments;
(c) digesting the product of step (b) with a second restriction endonuclease which is capable of cleaving said probe only at said second restriction site so as to produce labeled and unlabeled oligomer fragments;
(d) separating any labeled cleaved oligomer fragments from uncleaved oligomers;
and
(e) detecting the presence or absence of labeled fragments of particular base lengths which are formed on cleavage by the first and second restriction endonucleases.

3 more elements

Dependencies are inferred from the claim text.

Method for detection of polymorphic restriction sites and nucleic acid sequences

Life of this patent

Method for detection of polymorphic restriction sites and nucleic acid sequences

Life of this patent

Abstract

Claims28