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Methods of PCR-Based Detection of "Ultra Short" Nucleic Acid Sequences

a nucleic acid sequence and ultra-short technology, applied in the field of pcr-based detection of ultra-short nucleic acid sequences, can solve the problems of necrosis considered to be catastrophic metabolic failure, short specific nucleic acid biomarkers, and inflammation of surrounding cells, so as to reduce the degradation of nucleic acids, inhibit nuclease activity, and reduce the effect of nucleic acid degradation

Inactive Publication Date: 2010-03-18
TROVAGENE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]The nucleic acid degradation in the urine sample can be reduced. Reducing the nucleic acid degradation can include inhibiting nuclease activity by increased pH, increased salt concentration, heat inactivation, or by treating said urine sample with a compound selected from the group consisting of: ethylenediaminetetraacetic acid, guanidine-HCl guanidine isothiocyanate, N-lauroylsarcosine, or sodium dodecylsulphate. Preferably, the urine sample has been held in the bladder less than 12 hours.

Problems solved by technology

Necrosis is considered to be catastrophic metabolic failure resulting directly from severe molecular and / or structural damage and leads to inflammation and secondary damage to surrounding cells.
These specific nucleic acid biomarkers are often very short and their concentration in body fluids is usually low, especially if a test addresses an early stage of pregnancy or a disease.

Method used

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  • Methods of PCR-Based Detection of "Ultra Short" Nucleic Acid Sequences
  • Methods of PCR-Based Detection of "Ultra Short" Nucleic Acid Sequences
  • Methods of PCR-Based Detection of "Ultra Short" Nucleic Acid Sequences

Examples

Experimental program
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example 1

[0190]Example 1 shows the design of primers with internally located fluorophores for detection of ultra short DNA targets by PCR using fret-dependent fluorescence.

[0191]The specificity of a typical labeled-primer qPCR assay is determined solely by the specificity of the two primers, and therefore such an assay is not capable of distinguishing between templates having identical primer binding sites but different intervening sequences. To detect very short target fragments and overcome this limitation, novel labeled-primer assays were developed specific for targets in which the primer binding sites are either immediately adjacent to each other or even slightly overlapping, thus lacking any intervening sequences. Such amplicons are characterized by the pair of primer-derived sequences positioned in close proximity to each other in the double-stranded PCR product. In the assay, the mutual proximity of fluorescently labeled oligonucleotides was detected by Förster resonance energy transf...

example 2

[0200]Example 2 shows the design of primers with fluorophores located on 5′-end for detection of ultra short DNA targets by PCR using fret-dependent fluorescence

[0201]Compared to unlabeled primers, the use of primers labeled with fluorophores near their 3′ ends may lower the efficiency of the PCR, most likely due to reduction in polymerase processivity as it reads fluorophore-labeled bases of the primers incorporated into templates. To overcome this problem, another variant of the labeled-primer FRET qPCR scheme was developed. In this example, as shown in FIG. 7, the primers contain oligonucleotide tails at the 5′ ends of their target-binding sequences. In FIG. 7, T designates a template; P1 and P2 designate primers; D and A designate donor and acceptor fluorophores, respectively; black circles denote the positions of replication-blocking modifications. The tails are labeled at their 5′ ends with appropriate fluorophores, so that each double-stranded PCR product molecule carries a F...

example 3

[0203]Example 3 shows the design of single-tube PCR scheme for detection of ultra short DNA targets.

[0204]A novel qPCR scheme was developed that offers high target specificity by utilizing three sequence-specific components, including a TaqMan probe, yet allows for very short amplicons by means of a partial target recognition sequence overlap of the TaqMan probe with the sense (same-strand) target-specific primer. To accommodate the overlapping probe and primer, a novel 2-stage single-tube qPCR scheme was devised. The flow diagram provided in FIG. 8 shows the specific details and steps of the reaction. In FIG. 8, T designates a template; P1, P2, and P3 designate primers; IP1 and IP2 designate intermediate products; Pr designates TaqMan™ probe, dual-labeled with fluorophore F and quencher Q. In stage 1, the target DNA template T is amplified using primers P1 and P2, which map in very close proximity to each other on the target sequence, thus allowing for very short templates. Primer ...

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Abstract

The present invention provides highly sensitive methods used for diagnosing and monitoring various diseases and disorders by detecting and analyzing “ultra short” (20-50 base pair) nucleic acids obtained from bodily fluids.

Description

RELATED APPLICATIONS[0001]The present application claims priority to, and the benefit of, U.S. Provisional Application No. 61 / 135,364, filed Jul. 18, 2008. The contents of this application is herein incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention provides highly sensitive methods used for diagnosing and monitoring various diseases and disorders by detecting and analyzing “ultra short” (20-50 base pair) nucleic acids obtained from bodily fluids.BACKGROUND OF THE INVENTION[0003]Cell death is an essential event in the development and functioning of multicellular organisms. In adult organisms, cell death plays a complementary role to mitosis in the regulation of cell populations. The pathogenesis of numerous diseases involves failure of tissue homeostasis which is presumed to be linked with cytotoxic injury or loss of normal control of cell death.[0004]There exist two major types of cell death, necrosis and apoptosis, marked by different morph...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6846C12Q1/6851C12Q1/6886C12Q2525/204C12Q2523/308C12Q2565/101C12Q2525/186C12Q2525/207C12Q2525/161C12Q2600/106C12Q2600/156C12Q1/705C12Q2600/158
Inventor UMANSKY, SAMUIL R.MELKONYAN, HOVSEP S.OSSINA, NATALYASHEKHTMAN, EUGENE M.
Owner TROVAGENE
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