Cleavage of nucleic acids

Abstract

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

Description

[0001] This is a Continuation-In-Part of co-pending application Ser. No. 08 / 682,853, filed Jul. 12, 1996, which is a Continuation-In-Part of co-pending application Ser. No. 08 / 599,491, filed on Jan. 24, 1996.FIELD OF THE INVENTION [0002] The present invention relates to means for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5′ nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further provides novel methods and devices for the separation of nucleic acid molecules based by charge. BACKGROUND OF THE INVENTION [0003] The detection and characterization of specific nucleic acid ...

AI Technical Summary

Benefits of technology

"The present invention relates to a method for detecting specific nucleic acid sequences in a site-specific manner. This is achieved by using a cleavage structure that is specific to the nucleic acid sequence being detected. The cleavage structure includes a target nucleic acid, two oligonucleotides, and a third oligonucleotide. The two oligonucleotides are complementary to different regions of the target nucleic acid, and the third oligonucleotide has a specific sequence that matches the second region of the target nucleic acid. The invention is not limited by the length of the target nucleic acid or the nature of the oligonucleotides. The invention can be used for various purposes, including clinical diagnostic purposes."

Problems solved by technology

The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.

However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37° C.).

Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes.

In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield.

Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator.

The laboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting.

Indeed, PCR has yet to penetrate the clinical market in a significant way.

In addition, both methods require expensive equipment, capable of precise temperature cycling.

This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect (Kwok et al., Nucl.

Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification.

Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.

Traditional methods of direct detection including Northern and Southern blotting and RNase protection assays usually require the use of radioactivity and are not amenable to automation.

While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may be carried through sample preparation.

Requiring two oligonucleotides to bind to a target nucleic acid reduces the chance that false “positive” results will be produced due to the non-specific binding of a probe to the target.

This is in part because the DNA polymerase used in PCR can accommodate very large distances, measured in nucleotides, between the oligonucleotides and thus there is a large window in which non-specific binding of an oligonucleotide can lead to exponential amplification of inappropriate product.

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