Patent Application
20170002398
The present invention is in the field of molecular biology. Particularly, the present invention relates to the detection of nucleic acids, e.g. in amplification reactions such as polymerase chain reactions (PCR). The present invention is particularly useful in genotyping, quantitative PCR, real-time PCR and multiplex-PCR. The present invention particularly relates to dyes used in PCR.
Fluorescent dyes have a wide application in analytical chemistry, biochemistry and molecular biology, such as in DNA sequencing and during detection and amplification of nucleic acids. Fluorescent dyes absorb light of a specific wavelength (“excitation”) and re-emit energy at a different specific wavelength (“emission”). The fluorescent dyes are frequently used as molecular labels attached to probes or other biomolecules.
Multiplex polymerase chain reactions (multiplex PCR) is a PCR technique that enables amplification of two or more products in parallel in a single reaction tube. It is widely used in genotyping applications and different areas of molecular biology, e.g. in research, forensic and diagnostic applications, including human identification and paternity testing and for diagnosis of infectious diseases or chimerism analysis after allogeneic bone marrow transplantation. Multiplex PCR can also be used for qualitative and semi-quantitative gene expression analysis using cDNA as a starting template or in combination with reverse transcription with mRNA as starting material. The nucleic acids analyzed may for example originate from a variety of eukaryotic (human, animal or plant) and prokaryotic (bacterial) or viral sources.
Multiplex PCR is for example used for the simultaneous detection of multiple marker genes and/or their polymorphisms, e.g. short tandem repeats (STRs) or deletion insertion polymorphisms (DIPS or Indels). Detection of the amplification products and their genotyping is usually carried out by multiple color fluorescence detection after electrophorectic separation (e.g. capillary gel electrophoresis) in DNA sequencers. In real-time, quantitative multiplex PCR, the amplification of multiple target sequences can be monitored at the same time by simultaneous detection of fluorescence of different fluorescent dyes.
For the purpose of multiple colour fluorescence detection, at least one primer of each primer pair used during amplification is labeled by covalently bond fluorescent dye at is 5′-OH end or at an internal base.
To date, the simultaneous amplification of 8 to 16 or even more target sequences is possible. However, modern thermocyclers (for real-time PCR) and DNA sequencers used in multiplex PCR are adapted for the simultaneous detection of 3 to 5 different fluorescent dyes. Usually one of the fluorescent dyes is used as a label of a length standard when gel electrophoresis analysis is performed.
The apparatuses used for detection are often designed and calibrated for the use of combinations of very specific fluorescent dyes, i.e. in terms of excitation and detection wavelengths and filters used. The optical set-up of many devices encompasses the use of one Argon laser (488 nm) or a solid state diode (505 nm), a spectrograph, virtual filters, in combination with a CCD camera and algorithms for spectral calculation. This set-up severely limits the amount of dyes that may be used.
At the same time the identification of a dye in the spectral emission region 605 to 630 nm is difficult. These are difficult to excite by the known and used lasers. However the sensitivity in this range is the relevant factor for the overall sensitivity of the set-up. Hence, it would be desirable to have an ideal dye also in this spectral range.
The present invention relates to specific combinations of fluorescent dyes for the detection of fluorescently labeled nucleic acids.
The invention relates to a method for the simultaneous detection of at least four nucleic acids labeled with a covalently attached dye in a sample comprising the step of:
The invention also relates to a method for the simultaneous detection of at least four amplification products from an amplification reaction, comprising the steps of
And, the invention relates to a kit for multiplex PCR or PCR comprising at least four different fluorescent dyes suitable for the covalent attachment to nucleotides, primers, size markers or oligonucleotide probes used in multiplex PCR, wherein the four dyes may be selected from the six groups below:
The dye collection described herein for the first time provides for higher sensitivity, less cross talk between color channels and compatibility with numerous existing devices.
The present invention relates to specific combinations of fluorescent dyes for the detection of fluorescently labeled nucleic acids. The structures of 5-FAM (5-carboxyfluorescin) and 6-FAM (6-carboxyfluorescin) are illustrated in the figures. The structures of HEX (6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein) and ROX (5-carboxy-X-rhodamine) are illustrated in the figures. The structures of DY-530, DY-555, DY-556, DY-510XL, DY-632 and DY-520XL are illustrated in the figures. The structures of ATTO 532, ATTO 550 and ATTO 565 are illustrated in the figures. DY-530, DY-555, DY-556, DY-510XL, DY-632 and DY-520XL are products of Dyomics GmbH, Jena, Germany. ATTO 532, ATTO 550 and ATTO 565 are products of ATTO-TEC GMBH, Siegen, Germany. Chromeo™ 494 is a product of Active Motif, Carlsbad, Calif., US.
The invention relates to a method for the simultaneous detection of at least four nucleic acids labeled with a covalently attached dye in a sample comprising the step of:
In a preferred embodiment, the invention relates to a method for the simultaneous detection of at least six nucleic acids labeled with a covalently attached dye in a sample comprising the step of:
The invention also relates to a method for the simultaneous detection of at least four amplification products from an amplification reaction, comprising the steps of
In a preferred embodiment, the invention relates to a method for the simultaneous detection of at least six amplification products from an amplification reaction, comprising the steps of:
As outlined above, the invention relate to combinations of at least four different fluorescent dyes. In the context of the above described methods this means that when a size marker fluorescently labelled with one dye of the invention is used, at least three nucleic acid sequences (i.e. loci) can be amplified and detected, depending on whether a combination of four, five or even more different dyes is used. If no fluorescently labelled size marker is used, then at least four, five, or six sequences can be amplified and detected.
A “locus” in the context of the present invention is a part of the sequence of a nucleic acid, e.g. a chromosome, mRNA, plasmid, cosmid, DNA or RNA (of any origin, e.g. bacterial, viral, eukaryotic, prokaryotic, from plants, fungi or animals, e.g. of human origin) and the like, that is to be amplified and/or detected.
“Amplification products” herein are nucleic acids or oligonucleotides that are the product of an amplification reaction, e.g. of a polymerase chain reaction. They are for example defined by the primers used for amplification.
A “primer” herein refers to an oligonucleotide comprising a sequence that is complementary to a nucleic acid to be amplified or transcribed (“template”). During replication polymerases attach nucleotides to the 3′-OH end of the primer complementary to the respective nucleotides of the template.
An “oligonucleotide” herein refers to a stretch of nucleic acid, e.g. RNA or DNA, that comprises a sequence of two or more nucleotides, e.g. between 2 and 250 nucleotides, more preferably between 2 and 200, even more preferably between 2 and 100, even more preferably between 2 and 30, even more preferably between 2 and 25, even more preferably between 2 and 20, even more preferably between 5 and 25, and most preferably between 10 and 25 nucleotides.
In the first aspect of the method, during amplification detection may for example occur using fluorescently labelled oligonucleotide probes. Alternatively fluorescently labelled primers may be used. In another alternative, fluorescently labelled nucleotides may be used that are incorporated into the amplification products. In this case it is preferred that for every locus to be amplified or detected, the amplification is performed in a separate reaction tube. The amplification products may then be unified for detection. Thus, detection may in some cases occur during amplification, e.g. after or during each cycle of a polymerase chain reaction and/or after the complete amplification reaction.
In one embodiment of the first aspect of the method, the method additionally comprises the step of separating the amplification products by their size or molecular weight before detecting said amplification products. Separation by size or molecular weight can e.g. be performed using electrophoresis, e.g. gel electrophoresis, or chromatographical techniques.
The amplification reaction is preferably selected from the group comprising polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR), QP amplification and (thermostable) helicase dependent amplification ((t)HAD). More preferably the amplification reaction is a PCR. Most preferably, the amplification reaction is a multiplex PCR, i.e. the amplification of more than one target nucleic acid sequence in a single tube, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 loci (target nucleic acid sequences) may be amplified simultaneously.
As outlined above, the nucleic acid sequences (i.e. the loci) may be amplified simultaneously, e.g. in one reaction tube or separately, e.g. in different reaction tubes. It is preferred in the methods of the present invention that said loci are amplified simultaneously.
Preferably, the amplification reaction is a multiplex PCR.
Preferably, the reaction additionally comprises the step of separating the amplification products by their size or molecular weight before detecting said amplification products.
Preferably, the amplification reaction is real-time multiplex polymerase chain reaction and the method comprises the steps of
More preferably the amplification reaction is real-time multiplex polymerase chain reaction and the method comprises the steps of:
Here, one combination is preferred, i.e. 6-FAM, DY-530, ATT0565, ATT0550, DY510-XL, DY632 and Chromeo 494
Ideally, the first dye is 6-FAM, the second dye is ATT0550 the third dye is ATT0565, and the fourth dye is Chromeo 494.
If more than four dyes are used and these are preferably 6-FAM, DY-530, ATT0550, ATT0565, DY510-XL, DY632 and Chromeo 494.
If six dyes are used these are preferably 6-FAM, DY-530, ATT0550, ATT0565, DY632 and Chromeo 494.
If more than six dyes are used these are preferably selected from the group comprising 6-FAM, DY-530, ATT0550, ATT0565, DY510-XL, DY632 and Chromeo 494.
If more than six dyes are used these are preferably selected from the group consisting of 6-FAM, DY-530, ATT0550, ATT0565, DY510-XL, DY632 and Chromeo 494.
Preferably, at least one dye is covalently attached to an oligonucleotide size marker.
The invention also relates to a composition comprising:
In a preferred embodiment, the composition comprises:
Such a composition may be a pre-mix for a PCR reaction or a multiplex PCR reaction. It may comprise other components as a buffer or an enzyme.
In a further embodiment the invention relates to a kit for multiplex PCR or PCR comprising at least four different fluorescent dyes suitable for the covalent attachment to nucleotides, primers, size markers or oligonucleotide probes used in multiplex PCR, wherein the four dyes may be selected from the six groups below:
In a further embodiment the invention relates to a kit comprising different dyes, these being:
In a preferred embodiment, the invention relates to a kit for multiplex PCR or PCR comprising at least six different fluorescent dyes suitable for the covalent attachment to nucleotides, primers, size markers or oligonucleotide probes used in multiplex PCR, wherein the six dyes may be selected from the six groups below:
In one embodiment the amplification is an amplification of a forensic sample.
Object of the invention is the use of six defined fluorescent dyes that allow simultaneous detection of PCR products in capillary electrophoresis (CE). The challenge in choosing the fluorescent dyes is dependent on many factors. Forensic labs mainly use CE instruments from ABI (ThermoFisher). The optical setup of these devices consists of a single Argon-(488 nm) or “solide state” diode-laser (505 nm), a spectrograph, virtual filter sets in combination with CCD-cameras and algorithms for spectral calibration, which is entirely different from other CE-instruments or real-time PCR thermocyclers. This specific optical setup strongly limits compatibility of various dyes and thus the repertoire thereof. The aim is to achieve a minimal overlap of emission spectra of six different fluorescent dyes, a higher sensitivity, less crosstalk between the color channels, as well as compatibility with the ABI 3500 CE instrument. A panel of dyes were studied that meet these criteria while matching to the colors of a fluorescence dye combination, which can be used for DNA oligonucleotide labeling and simultaneous detection by DNA sequencing automates, consisting of 6-FAM, DY-530, ATT0550, DY510-XL and DY632.
Literature, patent, and internet searches revealed a panel of dyes that, according to the manufacturer, exhibit emission peaks within the defined spectral emission range (peak emission between 592 nm and 641 nm) for the sixth dye (Table 1).
Table 1 shows a list of candidates for the sixth dye as well as the source, feasibility of a CE-matrix with the dye, amount of background signal, and sensitivity. Six candidates could be calibrated successfully, of which five dyes yielded too high background signals or too low signal intensities. Only one dye (Chromeo 494) showed a very low background and strong signal intensity in combination with a five color dyeset, e.g. consisting of 6-FAM, DY-530, ATT0550, ATT0565 and DY632. A high sensitivity of the multiplex PCR assay is achieved by the low background noise and the strong signal intensity. This high sensitivity is a key factor, especially in forensic analyses coping with minuscule amounts of DNA samples.
The crosstalk into the other color channels is minimized by the matching spectrum of the Chromeo 494 with respect to a five color dyeset, e.g. consisting of 6-FAM, DY-530, ATT0550, ATT0565 and DY632. This reduces the occurrence of artefact peaks and facilitates the evaluation and interpretation of results.
This dye combination allows the amplification of more than 20 STR markers in a multiplex PCR and subsequent discrete separation by CE. This results in a distinct separation of single markers while retaining a reduced maximum length of PCR amplicons.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14152306.8 | Jan 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/050131 | 1/7/2015 | WO | 00 |
