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System For Multi Color Real Time PCR

a multi-color, real-time pcr technology, applied in the field of real-time pcr, can solve the problems of no respective successful application protocol published, no system disclosed for performing multi-pex experiments with a higher degree of complexity, and the design of appropriate fret hybridization probe sequences may sometimes be limited

Inactive Publication Date: 2011-06-23
ROCHE MOLECULAR SYST INC
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  • Abstract
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AI Technical Summary

Benefits of technology

[0089]Positioning of each reaction vessel (capillary) in a monitoring position is preferably characterized in that fluorescence excitation and monitoring are performed along the same axis of said reaction vessel. Such a principle enhances signalling due the phenomenon of total internal reflectance.
[0194]Furthermore, the Single Labeled Probes (SLP's) format for melting curve analysis (WO 02 / 14555) is applicable within a system according to the invention. This format is based on quenching or, respectively, de-quenching of the probe-label after hybridization to its target sequence. The format requires just one probe terminally labeled with a single dye. Compared to HybProbe or TaqMan formats, SLP's thus enable a significantly less expensive setup of assays for integrated analysis of Single Nucleotide Polymorphisms (SNP's). By reporting melting temperatures, different alleles of an SLP can unambigiously be distiguished in a single reaction. Multiplexing options are provided by using different reporter dyes.

Problems solved by technology

Yet, U.S. Pat. No. 6,015,674 does not disclose a system for performing multipex experiments with a higher degree of complexity.
Thus, although this instrument hardware at least theoretically has the capacity of performing multiplex experiments with up to four differently labeled hybridization probes within one reaction vessel, no respective successful application protocol has been published so far.
Yet, U.S. Pat. No. 6,369,893 does neither anticipate nor suggest any approach on how a multiplex experiment comprising multiple different probes each labeled with a different fluorescent entity needs to be designed.
Yet, the design of appropriate FRET Hybridization Probe sequences may sometimes be limited by the special characteristics of the target nucleic acid sequence to be detected.
Among other reasons, this may be due to lack of approriate instrumentation and, moreover, due to fact that the functionality of the FRET process of a specific FRET pair is interfered by other fluorecent compounds which are present in the same reaction mixture.
Yet, the utility of all of these instruments for multiplex detection up to now has been very limited due to the fact that attempts to establish real time multicolor multiplex assays with several (at least more than two) differently labeled probes with sufficient sensitivity and specificity have not been successful so far.

Method used

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  • System For Multi Color Real Time PCR
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  • System For Multi Color Real Time PCR

Examples

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Effect test

example 1

[0205]Quantitative real time PCR of Factor V DNA using different pairs of FRET hybridization probes labeled with different fluorescent compounds

[0206]For amplification of a Factor V DNA fragment, 4 different 100 μl real time PCR reaction mixtures were set up as follows:

106 copies of a plasmid containing Factor V gene 3 mM MgCl2500 nM eachprimers200 nMFRET 3′Hybridization Probeaccording toSeq. A200 nMFRET 5′Hybridization Probeaccording toSeq. B1, B2, B3 or B4, respectively.

[0207]In addition, PCR components from the LightCycler DNA Hyb Probes Kit (Roche Applied Science, Cat. No. 2158825) were used.

[0208]Primers and probes were used as follows:

Primer FactorV, forward:(Seq. ID. No: 1)5′-GAG AGA CAT CGC CTC TGG GCT A-3′ (22-mer)Primer FactorV, reverse:(Seq. ID. No: 2)5′-TGT TAT CAC ACT GGT GCT AA-3′ (20-mer)A: 3′Fluorescein labeled Hybridization Probe(SEq. Id. No: 3)5′-AAT ACC TGT ATT CCT CGC CTG TC-3′ (23-mer)(B1-B4: Seq. Id. NO: 4)B1: 5′ Red610 Hybridization Probe5′-AGG GAT CTG CTC TTA...

example 2

[0212]4-Color Melting Curve Analysis

[0213]A 479 by fragment of a specific plasmid containing a fragment of the human NAT-2 gene encoding N-Acetyl-Transferees isozyme was amplified with specific primers and detected by fluorescence, using 4 different specific pairs of FRET Hybridization

[0214]Probes. Whereas three probes were labeled at the 3′-end with Fluorescein, four detection probes were labeled 5′ either with Light- Cycler-Red 610, 640, 705 or Cy5 and modified at the 3′-end by phosphorylation. A schematic drawing of this experimental set up is shown in FIG. 6. As it can be deduced from the figure, there were four differently labeled FRET acceptor probes, but only three detection sites. This resulted in a competition of annealing of two detection probes.

[0215]Assay conditions were basically the same as disclosed in example 1 with the alteration that 3×106 copies of target DNA were used.

Primer forward:(Seq. Id. No: 5)5′-TGC CTT GCA TTT TCT GCT T-3′ (19 mer)Primer reverse:(Seq. Id. ...

example 3

[0219]Dual Color TaqMan Detection

[0220]A LightCycler instrument according to the best mode of the invention provided all means for sensitive detection of dual color TaqMan assays. The established TaqMan dye FAM and HEXs were well excited with the 470 nm LED and after using the instrument's color compensation function could differentially be identified in the 530 nm and 560 nm detection channels with high sensitivity. Transfer of dual color TaqMan assay protocols to the instrument according to the invention was simply performed by supplementing established assay conditions with BSA.

[0221]Different examples of successful dual color amplification and detection are shown in FIG. 8. FIG. 8a shows that in mono-color experiments using either FAM or HEX, a detection sensitivity of about 1 copy / μl for dual color TaqMan assays was obtained. (Only negative controls did not result in a significant increase in fluorescence after the PCR reaction). FIG. 8b shows a dual color experiment, wherein a...

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Abstract

The invention is directed to a system for performing multi-color real time PCR, comprising a flexible real time PCR instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixture which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

Description

FIELD OF INVENTION[0001]The present invention relates to the field of Real time PCR. In particular, the present invention is directed to a system for performing multiplex real time PCR.PRIOR ART BACKGROUND[0002]Amplification of DNA by polymerase chain reaction (PCR) is a technique fundamental to molecular biology. Nucleic acid analysis by PCR requires sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis. Th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12M1/34C12Q1/68
CPCC12Q1/6851G01N2021/6421C12Q2565/101C12Q2561/113C12Q2537/143
Inventor SAGNER, GREGORBECHLER, INGRIDBOLTE, JOCHENHEINDL, DIETERJOSEL, HANS-PETERGUTEKUNST, MARTINSEBL, RUDOLFMUELLER, CHRISTOPH
Owner ROCHE MOLECULAR SYST INC
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