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Primers and methods for the detection and discrimination of nucleic acids

a nucleic acid and primer technology, applied in the field of molecular biology, can solve the problems of requiring the sensitivity of such assays remains limited, and the inability to detect nucleic acids in the presence of two modified nucleotides, so as to reduce the background activity of the label, reduce the fluorescence background, and enhance the detection of target nucleic acid molecules

Inactive Publication Date: 2011-06-16
INVITROGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides oligonucleotides that can be modified at various locations and have specificity enhancing groups attached to them. These oligonucleotides can be used for detection and / or discrimination of target or template nucleic acid molecules by methods involving primer extension and hybridization. The oligonucleotides can also contain labels, such as fluorescent moieties, for detection and quantification of nucleic acid molecules. The specificity enhancing groups can include detectable groups, such as fluorescent groups, which undergo a detectable change upon extension of the oligonucleotide. Overall, the invention provides improved tools for detecting and analyzing target or template nucleic acid molecules."

Problems solved by technology

The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity.
Although the use of highly detectable labeled reagents can improve the sensitivity of nucleic acid detection assays, the sensitivity of such assays remains limited by practical problems which are largely related to non-specific reactions which increase the background signal produced in the absence of the nucleic acid the assay is designed to detect.
The major disadvantages of systems based on FRET are the cost of requiring the presence of two modified nucleotides in a detection oligonucleotide and the possibility that the efficiency of the quenching may not be sufficient to provide a usable difference in signal under a given set of assay conditions.
While this technology can be used to identify single nucleotide substitutions in a nucleic acid, it nonetheless suffers from some drawbacks in practical applications.
The difference in efficiency of amplification between the primers may not be sufficiently large to permit easily distinguishing between the normal nucleotide and the mutant nucleotide.
This problem requires careful selection of the number of amplification cycles and reaction conditions.
An additional problem with this methodology is presented by the detection step after the amplification.
The imposition of a separation step dramatically increases the time and expense required for conducting this type of analysis.
These methods suffer from the drawbacks discussed above.
None of these methodologies, describe or discuss real time detection of probes or primers, or changes in the fluorescence properties of a fluorescently labeled oligonucleotide upon hybridization or incorporation into amplified product.

Method used

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  • Primers and methods for the detection and discrimination of nucleic acids
  • Primers and methods for the detection and discrimination of nucleic acids
  • Primers and methods for the detection and discrimination of nucleic acids

Examples

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example 1

Preparation of Oligonucleotides

[0173]Oligonucleotides may be prepared using any known methodology. In some preferred embodiments, oligonucleotides may be synthesized on solid supports using commercially available technology. Oligodeoxynucleotides were synthesized using DNA synthesizer-8700 (Milligen / Biosearch). Fluorescent moieties may be incorporated into the oligonucleotides of the present invention using any conventional technology. For example, fluorescent labels may be incorporated into nucleoside phosphoramidites and directly incorporated into the oligodeoxynucleotides during automated chemical synthesis. In some preferred embodiments, the modified nucleotide may be a fluorescein-dT phosphoramidite (Glen Research Cat #10-1056) which may be inserted into designated position during chemical synthesis of oligonucleotide. 5′-fluorescein phosphoramidite (FAM) (Glen Research Cat # 10-5901) and 3′-TAMRA-CPG 500 (Glen Research cat #20-5910) were used to add the indicated labels to the...

example 2

PCR Targets and Conditions

[0183]Those skilled in the art will appreciate that any nucleic acid that can be amplified by PCR may be used in the practice of the present invention.

[0184]Examples of suitable nucleic acids include, but are not limited to, genomic DNAs, cDNAs and cloned PCR products. The practice of the present invention is not limited to use with DNA molecules. For example, mRNA molecules may be used as templates for an amplification reaction by first conducting a first strand synthesis reaction using techniques well known in the art. The present invention has been exemplified using cDNAs for IL4 and b-actin synthesized using total mRNA from the corresponding cells and SuperScript™ System for the First Strand cDNA Synthesis (Gibco BRL, cat #18089-011) according to the manufacturer's manual. IL4 and b-actin cDNAs were amplified and cloned into pTEPA plasmid according to Gibco BRL manual (cat #10156-016).

[0185]The selection of suitable PCR conditions is within the purview ...

example 3

Detection of Nucleic Acids

[0186]Nucleic acids may be detected by any conventional technology. In some preferred embodiments, the nucleic acid to be detected may be a PCR product and may be detected either by agarose gel electrophoresis or by homogeneous fluorescence detection method as described in U.S. provisional patent application Ser. No. 60 / 139,890, filed Jun. 22, 1999. In this method fluorescent signal is generated upon the incorporation of the specifically labeled primer into the PCR product. The method does not require the presence of any specific quenching moiety or detection oligonucleotide. In some preferred embodiments, the detection oligonucleotides are capable of forming a hairpin structure and are labeled with fluorescein attached close to the 3′-end.

[0187]The fluorescent measurements were performed in the PCR reaction buffer using on ABI PRIZM™7700 Sequence Detector, fluorescent plate reader (TECAN) or Kodak EDAS Digital Camera. Excitation / emission wavelengths were 4...

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Abstract

The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application is related to U.S. Provisional Patent Application Nos. 60 / 139,890, filed Jun. 22, 1999, and 60 / 175,959, filed Jan. 13, 2000, both of which are specifically incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to the field of molecular biology. In particular, the present invention relates to novel primers for use in the detection and discrimination of nucleic acids. The novel primers of the present invention will find broad applicability in the field of molecular biology and, in particular, in the detection of products in nucleic acid amplification reactions and in the discrimination between alleles of a given target gene.[0004]2. Related Art[0005]Assays capable of detecting and quantifying the presence of a particular nucleic acid molecule in a sample are of substantial importance in forensics, medicine, epidemiology and pub...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/00G01N33/53C12N15/09G01N33/566G01N33/58G01N37/00
CPCC12Q1/6816C12Q1/6818C12Q1/6827C12Q1/6832C12Q1/6851C12Q1/6858C12Q2565/107C12Q2563/107C12Q2561/12C12Q2561/119C12Q2525/301C12Q2525/101
Inventor NAZARENKO, IRINARASHTCHIAN, AYOUB
Owner INVITROGEN
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