Oligonucleotides and assemblies thereof useful in the detection of the presence or absence of target nucleic acid sequences in a sample

Abstract

Oligonucoleotides useful in the detection of a nucleic acid target sequence in a sample.

Description

[0001] This is a continuation-in-part of U.S. patent application Ser. No. 09 / 449,545, filed Nov. 29, 1999.FIELD AND BACKGROUND OF THE INVENTION[0002] The present invention relates to oligonucleotide probes and methods for the detection of target nucleic acid sequences in a sample. More particularly, the present invention relates to oligonucleotide probes which are internally cleavable when hybridized to target nucleic acid sequences, such cleavage leading to both signal generation and amplification by recycling.[0003] The identification of a target nucleic acid sequence is of great importance in both biological research and medical diagnostics. Detection of a target sequence can be used to identify and / or type a specific DNA or RNA molecule and to uncover mutations.[0004] Numerous methods and techniques exist in the art with which detection and / or identification of a target sequence can be effected. For example, polynucleotide sequencing methods can be used to determine the nucleoti...

AI Technical Summary

Benefits of technology

[0022] According to still further features in the described preferred embodiments the first, second, third and fourth regions of the oligonucleotide or assembly of oligonucleotides are further selected such that following cleavage of the nucleic acid cleaving agent recognition sequence, the first and second regions dissociate from the target nucleic acid sequence, thereby enabling recycling of the target nucleic acid sequence.

[0037] The present invention successfully addresses the shortcomings of the presently known configurations by providing oligonucleotides or assemblies of oligonucleotides which enable the simple and efficient detection of target nucleic acid sequences while reducing the reaction order or the background signal generation.

Problems solved by technology

However, the utility of linear oligonucleotide probes is frequently limited by their poor binding stability and selectivity.

The limitation of this approach is that no signal amplification is enabled, resulting in inability of detecting low target concentrations.

The limitation of this approach is that no signal amplification is enabled, resulting in inability of detecting low target concentrations.

Although the above mentioned methods are less complicated to perform than simple oligonucleotide probe detection methods such as that described by Sambrook et al. in which oligonucleotide probes are used to target nucleic acids that are immobilized onto a filter or membrane, some limitations still apply.

For example, a method which is simple to perform such as that described by Livak et al. can yield false positive results since hybridization to non-target sequences will also yield, in some cases, a positive result.

Methods which are aimed at producing more accurate results are oftentimes more complicated to perform.

However, there are cases in which the "universal" third and fourth regions can not be used with a target specific first and second regions.

Although these oligonucleotide probe configurations can be utilized for detection of target nucleic acid sequences they still suffer from several inherent limitations, such as, for example, low signal generation and template independent cleavage and signal generation.

Second, at least some of the oligonucleotides must be synthesized with target hybridizing regions which are long enough to allow hybridization but yet short enough to dissociate from target following cleavage of the stem and to minimize overall complexity of the molecule and as such the chances of undesirable secondary structures formation.

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