Cleavage of nucleic acids

Abstract

The present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Enzymes, including 5′ nucleases and 3′ exonucleases, are used to detect and identify nucleic acids derived from microorganisms. Methods are provided which allow for the detection and identification of bacterial and viral pathogens in a sample.

Description

[0001] The present application is a Continuation of U.S. application Ser. No. 09 / 941,193, filed Aug. 28, 2001, which is a Divisional application of U.S. App. Serial. No. 09 / 655,378, filed Sep. 5, 2000, now U.S. Pat. No. 6,673,616, which is a Continuation of U.S. application Ser. No. 08 / 520,946, filed Aug. 30, 1995, now U.S. Pat. No. 6,372,424, which is a Continuation-in-Part application of U.S. application Ser. No. 08 / 484,956, filed Jun. 7, 1995, now U.S. Pat. No. 5,843,654, which is a Continuation-in-Part of U.S. App. Serial. No. 08 / 402,601, filed Mar. 9, 1995, now abandoned, which is a Continuation-in-Part of U.S. application Ser. No. 08 / 337,164, filed Nov. 11, 1994, now abandoned, which is a Continuation-in-Part of U.S. application Ser. No. 08 / 254,359, filed Jun. 6, 1994, now U.S. Pat. No. 5,614,402, which is a Continuation-In-Part Application of U.S. application Ser. No. 08 / 073,384, filed Jun. 4, 1993, now U.S. Pat. No. 5,541,311, which is a Continuation-In-Part of U.S. applicat...

AI Technical Summary

Benefits of technology

[0061] By the term “genetic fingerprint” it is meant that changes in the sequence of the nucleic acid (e.g., a deletion, insertion or a single point substitution) alter the structures formed, thus changing the banding pattern (i.e., the “fingerprint” or “bar code”) to reflect the difference in the sequence, allowing rapid detection and identification of variants.

[0062] The methods of the present invention allow for simultan...

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 carried through sample preparation.

However, specialized equipment and highly trained personnel are required, and the method is too labor-intense and...

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