The present invention provides an asymmetric PCR method, the COMplementary-Primer-Asymmetric (COMPAS)-PCR, and specific methods for detecting, identifying or monitoring salmonid species. The present invention also encompasses oligonucleotide primers corresponding to species specific sequences. The use of the methods and primers are also aspects of the present invention together with kits comprising said primers.
Since the Polymerase Chain Reaction (PCR) was invented by Kary Mullis in the mid-80s [1], Nucleic Acid (NA) amplification techniques have had an unprecedented development for molecular biology applications. A contributing factor to this success is its flexibility with the development of several modifications which expands the technical capabilities of PCR. In particular several methods have been developed for the detection of point mutations such as the Amplification Refractory Mutation System (ARMS) [2] and variants such as the PCR Amplification of Specific Allels (PASA) [3, 4], bidirectional-PASA [5] or Mistmatch Amplification Mutation Assay (MAMA) [6], Taq-MAMA [7] and Melt-MAMA [8]. Point mutations, also called Single Nucleotide Polymorphism (SNP), constitute differentiating genetic information which may be relevant in various contexts spanning from medical applications for disease diagnostics to population genetics and species identification. As the amount of PCR application increases, limitations inherent to DNA chemistry may become more challenging. For instance primer complementarity leading to primer dimer formation, is a limiting factor for the design of generic PCR.
It is believed that the asymmetric PCR method according to the present invention will alleviate PCR limitations due to primer complementarity incompatibility. Said method was further applied for the development of a PCR assay for direct repeat sequences.
NA technology can be characterized as noninvasive since very little subject sample is required for obtaining sufficient NA for analysis. The fish farming industry is in need for such noninvasive methods for the identification of salmonid species and in particular Salmo solar (Atlantic salmon) and Salmo trutta (brown trout) and their hybrids. Methods for species determination and inter-species hybridization between different salmonid fish species is an important tool in ecological studies, and when assessing the impact of aquaculture escapees on indigenous populations. Ecological studies have shown that inter species hybridization can severely impact population size and viability estimates. Morphological discrimination between hybrids and parental species is in many cases difficult (e.g. S. salar X S. trutta), and the behavior of the hybrids may be different from that of the parental species [9].
Identification of fish species by PCR has previously been addressed and solutions have been suggested. However, when selecting available methods for identification of salmonid species as e.g. in References [10-14], the method published by A. M. Pendas et al., (Chromosomal mapping and nucleotide sequence of two tandem repeats of Atlantic salmon 5 S rDNA Cytogenet Cell genet 67:31-36 (1994)) [14] seemed to give the best result among the methods tested. The inventor surprisingly discovered that said method was not reliable for the above mentioned purpose as the identification of the species Salmo salar and Salmo trutta were not distinguished satisfactorily using the PCR method and primers described in [14]. It is believed that the present invention provides a robust simple PCR method capable of specifically identify Salmo salar and preferably differentiate between Salmo Salar, Salmo trutta and their hybrids. The inventor tested the Salmo-A & B PCR method by A. M. Pendas et al.; the primers were originally designed based on the 5S rDNA sequence of the rainbow trout (Salmo gairdnerii, renamed Oncorhynchus mykiss) [15] and used on Salmo trutta and Salmo salar among other fish species [16]. These primers amplify 118 bp of the 120 bp coding sequence of the 5S rDNA together with the associated variable non coding sequence. The amplification product(s) varies in length and were used to differentiate close species. Two loci were originally found to be amplified for Salmo salar, one major product about 255 bp and a minor product around 525 bp. These results are corroborated by the 2 published sequences for Salmo salar gb S73107.1 & gb S73106.1. Similarly, Salmo trutta also showed a double band pattern but with longer products both for the major and minor loci [14]. Hybrids between Salmo salar and Salmo trutta were found to produce both product types for the major loci around 255 bp and longer respectively [16]. No product(s) difference between Salmo salar and for Salmo trutta was detected by the inventor using the method of A. M. Pendas et al., to differentiate Salmo salar from Salmo trutta and their hybrids. However, the genetic target of said PCR method, the 5S-rDNA, was used to develop the novel and inventive method together with specific oligonucleotide primers and COMPAS PCR method of the present invention.
The present invention encompasses in a first aspect a method of asymmetric PCR comprising:
A second aspect of the present invention comprises a method for detecting, identification or monitoring salmonid species comprising:
In a third aspect the present invention provides oligonucleotide primers, selected from the oligonucleotides of Tables 1 and 2, or oligonucleotides with complementary sequences or functional equivalent sequences.
Further it is provided in a fourth aspect a kit for detecting and identification of salmonid species, comprising a collection of oligonucleotide primer pairs selected from Tables 1 and 2 in any combinations or complementary sequences thereof, capable of detecting salmonid species by the method of the present invention.
Finally in further aspects the present invention comprises use of the methods of the present invention. Use of oligonucleotide primers of the present invention or use of a kit for detecting and identification of salmonid species in accordance with the present invention.
Preferred embodiments are set forth in the dependent claims and in the detailed description of the invention
It is an object for the present invention to provide an asymmetric PCR method and non-invasive methods applicable for example for identification of salmonid species, preferably Salmo salar and Salmo trutta and their hybrids using almost complementary primers targeting direct tandem repeats. The present invention also encompasses oligonucleotide primers corresponding to species specific sequences or single nucleotide polymorphism (SNP). The use of the method and primers are also an aspect of the present invention together with kits comprising said primers. In order to unlock complementary primers for target product amplification, an asymmetric PCR method was developed by decreasing either the forward or the reverse primer concentration until optimal PCR amplification is reached. As shown in
In order to identify nucleotide sequences suitable as a target for discerning between the species, the inventor made a structural study of the 5 S-rDNA tandem direct repeats and noticed that any single section of the tandem direct repeat, covering a length typical for primers (i.e. 20 bp), would be appropriate to design complementary primers structurally covering one or more products depending on the tandem direct repeat number (see
To favor priming to the target DNA contra priming to the reverse primer, the inventor increased the forward 3′ primer end by 2 nucleotides which happened to be GG (strong priming). Similarly the 3′ end of the reverse primer was extended, to favor its priming to the target contra priming to the reverse primer.
The presence of genetic variations and its adequate exploitation is the key for developing a specific test. As published sequences did not provide appropriate genetic variation information for S. salar and S. trutta, the inventor systematically tested out reverse primers, incrementing by 1 nucleotide at the 3′ end. This explored one by one the possibility for SNP. As a basis for the testing, the reverse primer was chosen, as this primer extended in the non-transcribed sequence (NTS), which is more prone to variations than the coding sequence in which the forward sequence was extending (See
The method is not restricted to identification of fish species. Using almost fully complementary primers targeting the same sequence may also be applicable to any organism with tandem direct repeats DNA motifs of interest as target sequences. For example the 5S r-DNA tandem direct repeats is in essence found in all eukaryotic cells [21, 22] and could therefore be further used to develop specific complementary-primer assays for other taxon and species than salmonids. Further, the method will be useful for developing assays using primers targeting distinct sequences but generating primer-dimers due to part complementarity.
Accordingly a first aspect of the present invention relates to a method of asymmetric PCR comprising:
In one or more embodiments the highly complementary primers may have a common overlapping DNA target sequence, said sequence may be direct tandem repeats, the direct tandem repeats target may be in the 5S-rDNA region. Other regions may however also be an option.
As used herein “complementary primers” refers to primers that are complementary to each other and will under previously known conditions bind to each other to form primer-dimers.
A second aspect of the present invention relates to a method for detecting, identification or monitoring salmonid species comprising:
As used herein “salmonid” refers to a family of ray-finned fish. It includes salmon, trout, chars, freshwater whitefishes and graylings. The Atlantic salmon and trout of genus Salmo give the family and order their names.
In a further embodiment the determination of the species may be performed by a melting curve analysis of the PCR product or by electrophoresis analysis. Also other methods for determination of the PCR product may be employed.
In one or more embodiments the forward primer may be extended in the 3′ end to favor priming to the target and not to the reverse primer. The reverse primer may be extended in the 3′ end to favor priming to the target and not to the forward primer. The reverse or the forward primer may have a SNP at its 3′ end. Said primer may also be slipping out to the “right” in the 5S-rDNA coding sequence out of the complementary area with the reverse primer. Further the reverse primer may be extended in the 5′ end. The 3′ forward primer may be extended by 4, preferably by 3, more preferably by 2 nucleotides.
In a further embodiment primers in the primer pair (s) are oligonucleotides each having a length of about 12 to about 30, preferably about 20 bp.
In a further embodiment the complementary set of primers may be selected from a set of primer pair (s), wherein the forward primer may be selected from Table 1 or a complementary sequence thereof and the reverse primer may be selected from Table 2 or a complementary sequence thereof or any combinations thereof.
The reverse primer may be locked at its 3′ end at “+3” in the non-coding sequence, out of the complementary forward primer area, as the specificity lies in precise positioning of this SNP. At the other end, in 5′, the reverse primer may be shorter or longer (depends on the forward primer).
In one embodiment the salmonid of the present method may be Salmo trutta, Salmo solar and hybrids thereof. The method may however, be applicable to other salmonid species, other fish species or in fact any organism with tandem repeats DNA motifs.
In a third aspect the present invention comprises oligonucleotide primers, which may be selected from the oligonucleotides of Tables 1 and 2 or any combinations thereof, or oligonucleotides with complementary sequences or functional equivalent sequences. The use of the oligonucleotides in a product, e.g a kit form furthers aspects of the present invention.
In a fourth aspect the present invention provides a kit for detecting and identification of salmonid species, comprising a collection of oligonucleotide primer pairs selected from Tables 1 and 2, in any combinations, or complementary sequences thereof, capable of detecting salmonid species by the method of the present invention.
Finally in further aspects the present invention comprises use of the methods of the present invention, use the oligonucleotide primers of the present invention or use of the kits provided from the present invention.
Having now fully described the present invention in some detail by way of illustration and example for purpose of clarity of understanding, it will be obvious to one of ordinary skill in the art that same can be performed by modifying or changing the invention by using a wide and equivalent range of conditions and other parameters thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
Finn clips were collected from 1 S. trutta and 1 S. salar fish individuals for analysis. The samples were conserved in 98% EtOH prior to DNA extraction that was performed using mechanical and chemical methods for releasing PCR-grade DNA. Subsequent DNA measurement was performed using a nanodrop instrument (Thermo Scientific) and all samples were diluted in water to achieve a final concentration of 4 ng/μl. Samples (2.5 μl) were amplified in 25 μl final reaction mixtures using an ABI 7500 qPCR machine (Life technologies, Applied Biosystems). The mixture contained 12.5 μl MESA Blue qPCR MasterMix (Eurogentec), 0.6 μM reverse primer 5SNTS-23R+3, the forward primer 5SNTS-23F concentration was successively tested using: 0.6, 0.4, 0.2, 0.1 and 0.05 μM, the reaction was completed with distilled water, final volume of 25 μl. The 3-step PCR conditions consisted of 5 min activation at 95° C. followed by 30 cycles of 95° C. for 20 s, 62° C. for 30 s and 72° C. for 60 s. Real time amplification curves are shown in
Finn clips were collected from 4 S. trutta and 4 S. salar fish individuals for analysis. The samples were conserved in 98% EtOH prior to DNA extraction that was performed using mechanical and chemical methods for releasing PCR-grade DNA. Subsequent DNA measurement was performed using a nanodrop instrument (Thermo Scientific) and all samples were diluted in water to achieve a final concentration of 4 ng/μl. Samples (2.5 μl) were amplified in 25 μl final reaction mixtures using an ABI 7500 qPCR machine (Life technologies, Applied Biosystems). The mixture contained 12.5 μl MESA Blue qPCR MasterMix (Eurogentec), 0.01 μM forward primer 5SNTS-23F, 0.6 μM reverse primer 5SNTS-23R+3 completed with distilled water. The 2-step PCR conditions consisted of 5 min activation at 95° C. followed by 30 cycles of 95° C. for 20 s and 72° C. for 30 s. Products were visualized on 1,2% agarose gels (Fermentas) stained with SybrGreen (See
Number | Date | Country | Kind |
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20130468 | Apr 2013 | NO | national |
Filing Document | Filing Date | Country | Kind |
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PCT/NO2014/050049 | 4/7/2014 | WO | 00 |