CA2821014A1 - Sex discernment by real-time pcr in coho salmon - Google Patents
Sex discernment by real-time pcr in coho salmon Download PDFInfo
- Publication number
- CA2821014A1 CA2821014A1 CA2821014A CA2821014A CA2821014A1 CA 2821014 A1 CA2821014 A1 CA 2821014A1 CA 2821014 A CA2821014 A CA 2821014A CA 2821014 A CA2821014 A CA 2821014A CA 2821014 A1 CA2821014 A1 CA 2821014A1
- Authority
- CA
- Canada
- Prior art keywords
- coho salmon
- sex
- real
- pcr
- primers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 241000277338 Oncorhynchus kisutch Species 0.000 title claims abstract description 39
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 17
- 238000003752 polymerase chain reaction Methods 0.000 claims abstract description 15
- 239000000203 mixture Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims description 16
- 102000053602 DNA Human genes 0.000 claims description 16
- 230000003321 amplification Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 210000003205 muscle Anatomy 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000000523 sample Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 4
- 230000020509 sex determination Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000277275 Oncorhynchus mykiss Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000277331 Salmonidae Species 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000019688 fish Nutrition 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241001280377 Oncorhynchus tshawytscha Species 0.000 description 1
- 210000002593 Y chromosome Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 210000003765 sex chromosome Anatomy 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A sex discernment method that uses real-time polymerase chain reaction (PCR) to discern the sex of Coho salmon (Oncorhynchus kisutch) comprises the following steps: extracting and amplifying Coho salmon DNA by PCR, using the fluorescent probe pCsex1 (FAM) 5'-CCTGTGTATACTTGGGAGCGGTAAGCC-3' and a mixture of primers, CSex2s: 5'-CATTATATTTGGCTGAAGAGTCAGTG-3' and CSex2r: 5'-GGAACAGATGGAGCGGTCTTC-3'; identifying the presence of the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTGAATTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTATACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCTCCATCTGTTCC, as a fluorescence signal, the presence of the fluorescence signal indicating male Coho salmon and the absence of the fluorescence signal indicating female Coho salmon. The mixture of primers and the kit comprising the mixture of primers and the fluorescent probe are also disclosed.
Description
SEX DISCERNMENT BY REAL-TIME PCR IN COHO SALMON
Field of the Invention The present invention relates to a method for discerning sex by the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), related primers and kit.
State of the Art The real-time polymerase chain reaction (PCR) technique consists of the in vitro amplification of a specific sequence of interest, whose amplification generates a fluorescent signal detectable by the equipment. In function of the signal's behaviour, we can establish the abundance of the amplified sequence. Therefore, we can have two results with this technique, the first one consists of establishing the presence or absence of a sequence of interest in a sample being analyzed, and secondly, we can establish a kind of relative quantification of this sequence in the sample.
In several species, wherein sex determination is given by heterogametic chromosomes, in which in one of the chromosomes there exists a gene containing the information for determining sex, in the case of the human species this gene is designated as sry, which is located in chromosome Y.
In salmonid species, it is also known that sex determination is given by heterogametic chromosomes. However, a Sry-type gene as in humans has not been found to this date.
Field of the Invention The present invention relates to a method for discerning sex by the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), related primers and kit.
State of the Art The real-time polymerase chain reaction (PCR) technique consists of the in vitro amplification of a specific sequence of interest, whose amplification generates a fluorescent signal detectable by the equipment. In function of the signal's behaviour, we can establish the abundance of the amplified sequence. Therefore, we can have two results with this technique, the first one consists of establishing the presence or absence of a sequence of interest in a sample being analyzed, and secondly, we can establish a kind of relative quantification of this sequence in the sample.
In several species, wherein sex determination is given by heterogametic chromosomes, in which in one of the chromosomes there exists a gene containing the information for determining sex, in the case of the human species this gene is designated as sry, which is located in chromosome Y.
In salmonid species, it is also known that sex determination is given by heterogametic chromosomes. However, a Sry-type gene as in humans has not been found to this date.
In the Crossing Programs for coho salmon, in culture, it is only required to have a proportion of 1/3 males relative to females for the generation of a sufficient number of reproductors.
One object of the present invention is to provide a method for discerning sex in coho salmon which would allow to confirm the existence of a sufficient amount of males for crossings, rather than an excess or reduction of same.
In addition, the method of the present invention allows to evaluate "Neomale"
production systems in coho salmon production systems. Neomale fish are females which through various methodologies revert to a male phenotype. However, at the genetic sequence level, they do not exhibit changes, these only occur at the phenotypic level. Therefore, this real-time PCR
methodology may be applied to evaluate the efficiency of Neomale production in a Genetic Program in coho salmon rearing.
Brief Description of the Invention Based on bioinformatic analyses, a putative genomic region was identified which would be present only in males of the coho salmon species, which for convenience we will designate as "Cohoseq" in the text, and comprises the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTAA
TTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTAT
ACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCTC
CATCTGTTCC; to evaluate the presence of this sequence in a biological sample, a real-time PCR application was developed which allows to evaluate the presence of this sequence in the sample. Up to date, there does not exist in the literature any methodology for conducting sex determination in coho salmon which incorporates the use of real-time PCR.
The present invention relates to a method for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), which comprises the following steps:
a) extracting deoxyribonucleic acid (DNA) from coho salmon either from biological or solid or semisolid matrices, such as muscle, fin, gill or the like, or fluids, such as total blood, plasma and serum;
optionally storing the DNA eluate obtained in step (a) at 4 C;
b) amplifying the DNA eluate from step (b) by the use of polymerase chain reaction (PCR), in real-time format, using the fluorescent probe pCsex 1 which corresponds to the sequence (FAM) 5 "-CCTGTGTATACTTGGGAGCGGTAAGCC-3" and a mixture of primers comprising the CSex2s primer: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and the CSex2r primer: 5 '-GGAACAGATGGAGCGGTCTTC-3 ';
c) identifying the presence of the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTGA
ATTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTA
TACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCT
CCATCTGTTCC, as a fluorescence signal, in the amplification resulting from step (c), assigning to the presence of said fluorescence signal the determination of male sex in coho salmon, whose DNA was amplified, and to the absence of said fluorescence signal, the determination of female sex in coho salmon, whose DNA was amplified.
The present invention also relates to a mixture of primers for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), which comprises the synthetic sequences CSex2s: 5 .-CATTATATTTGGCTGAAGAGTCAGTG-3 and CSex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 In addition, the invention comprises a kit for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), which comprises:
a) a mixture of primers comprising the synthetic sequences CSex2s: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and C Sex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 ;
b) a fluorescent probe comprising the sequence:
pCsex 1 (FAM) 5 '-CCTGTGTATACTTGGGAGCGGTAAGCC-3 "(BHQ).
Brief Description of the Figures Figure 1: Amplification curve by real-time PCR in sex determination of coho salmon, the curve represents the presence of genetic sequence only in males.
Example, methodology The extraction of genetic material can be performed from multiple sources. In the case of using solid or semisolid biological matrices, such as muscle, fin, gill, etc., the sample is placed inside a bag and is macerated with a rubber hammer. Subsequently, phosphate buffered saline (PBS) buffer is added enough to reach a suitable turbidity (equivalent to No. 5 of the McFarland scale).
Samples corresponding to fluids (total blood, plasma and serum) are directly treated.
Subsequently, the DNA (deoxyribonucleic acid) extraction is performed, for example, with a ' = CA 02821014 2013-06-10 commercial kit. For this example, the protocol of the commercial kit QIAampODNA Blood Mini Kit will be used (Qiagen; catalog No. 51104X), wherein the description of the methodology will mention solutions whose abbreviations correspond to the original ones disclosed in the commercial kit (for example, AL buffer). To begin with the DNA extraction, 200 ml of sample (homogenate or fluid, whichever the case may be) are taken and introduced into an Eppendorf tube which has not been previously used, 20 ul Proteinase K (Invitrogen) are added and it is gently homogenized to be incubated during 30 minutes at 60 C. Subsequently, 200 ul of AL
buffer are added to the sample in the tube. Homogenize by vollexing for 15 seconds and incubate at 56 C for 12 min. Briefly centrifuge at low revolution speed in order to avoid leaving drops (3000 rpm for 10 sec) on the edges. Add 200 ul of absolute ethanol (96-100%) and homogenize by vortexing for 15 seconds. Carefully add the homogenate to a column and centrifuge at 10000 x g for 1 min. Subsequently, change the collection tube to another collection tube which has not been previously used. Add 500 ul of AW1 buffer, incubate for 1 min in the column and centrifuge at 10000 x g for 1 min. Change the collection tube to another collection tube which has not been previously used. Add 500 ul of AW2 buffer, incubate for 1 min in the column and centrifuge at maximum speed for 3 min. Place the column in another collection tube which has not been previously used and add 50 ul of molecular biology grade water, and incubate at room temperature for 5 min. Finally, centrifuge at maximum speed for 1 min and store the eluate (DNA; deoxyribonucleic acid) at 4 C before use thereof.
= = CA 02821014 2013-06-10 , In order to carry out the amplification phase of the genetic material, PCR in real-time format is used. For this part of the assay, for example, a commercial kit (Express qPCR
Supermix Universal Kit (Invitrogen)) is used; for this purpose, a number of reactions based on the number of samples to be analyzed will be prepared. For example, for preparing 10 amplification reactions, prepare the microplates corresponding to this number of reactions and prepare the master mix as described in the kit; the following table shows the volumes of each component for preparing the master mix:
Master Mix 10X (u1) Express qPCR SuperMix with Premixed ROX 10 100 Fluorescent probe pCsexl (1)(O.3 uM) 10 Mixture of primers (2) (0.9 uM) 10 Nuclease-free water 70 (I) pCsex 1 (FAM, 5'-CCTGTGTATACTTGGGAGCGGTAAGCC-3' (BHQ).
FAM: 6-carboxy-fluorescein (fluorophore) BHQ: Black Hole Quencher (Quencher; fluorescent silencer) =
Nuclease-free water: water that is free of nucleases which are enzymes that degrade genetic material.
(2) Primer sequences:
C Sex2s: 5'-CATTATATTTGGCTGAAGAGTCAGTG-3' and CSex2r: 5'-GGAACAGATGGAGCGGTCTTC-3'.
Subsequently, 19 ul of the master mix are placed in each of the microtubes and 1 ul of the previously extracted DNA sample is introduced into each one. Subsequently, place the tubes in a real-time thermocycler with the following thermal profile or schedule:
1 cycle: 95 C, 20 seconds 40 cycles: 95 C, 10 seconds 55 C, 15 sec*, fluorescence reading 1 cycle: 25 C, 30 seconds At the end of the amplification schedule, the results are interpreted as detailed below.
In each microtube in which a real-time PCR reaction was conducted for discerning sex in a coho salmon sample, two events may occur. First, that a fluorescence signal is generated, which is interpreted as the presence of the Cohoseq sequence, which in turn is interpreted as male sex of the salmonid since the female should not have this region. Second, that a fluorescence signal is not generated, which is interpreted as female sex since the Cohoseq sequence is not found, and therefore, there is no substrate for the generation of a signal. Figure 1 shows an analysis of some samples, it can be observed that there are several amplification events. The amplifying curves correspond to samples that were identified as male coho salmon, while the samples that did not amplify correspond to samples that were identified as female coho salmon.
In parallel, 60 coho salmon samples were analyzed, 30 female and 30 male, fish whose sex was previously established by visual inspection since they were adult specimens.
Then, the samples were submitted to a real-time PCR analysis, obtaining 100% agreement of the results obtained with the prior information related to the sex of the samples, which was previously established by visual inspection.
Bibliography BruneIli J.P., Wertzler K.W., Sundin K and Thorgaard G.H. 2008. Y-specific sequences and polymorphisms in rainbow trout and Chinook salmon. Genome 51, 739-748.
Felip A, Fujiwara A., Young W.P., Wheeler PA, Noakes M., Phillips R.B and Thorgaard G.H.
2004. Polymorphism and differentiation of rainbow trout Y chromosomes. Genome 47, 1105-1113 .
Iturra P., Lam N., De la Fuente M., Vergara N and Medrano J.F. 2001.
Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH).
Genetica 111, 125-131.
One object of the present invention is to provide a method for discerning sex in coho salmon which would allow to confirm the existence of a sufficient amount of males for crossings, rather than an excess or reduction of same.
In addition, the method of the present invention allows to evaluate "Neomale"
production systems in coho salmon production systems. Neomale fish are females which through various methodologies revert to a male phenotype. However, at the genetic sequence level, they do not exhibit changes, these only occur at the phenotypic level. Therefore, this real-time PCR
methodology may be applied to evaluate the efficiency of Neomale production in a Genetic Program in coho salmon rearing.
Brief Description of the Invention Based on bioinformatic analyses, a putative genomic region was identified which would be present only in males of the coho salmon species, which for convenience we will designate as "Cohoseq" in the text, and comprises the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTAA
TTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTAT
ACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCTC
CATCTGTTCC; to evaluate the presence of this sequence in a biological sample, a real-time PCR application was developed which allows to evaluate the presence of this sequence in the sample. Up to date, there does not exist in the literature any methodology for conducting sex determination in coho salmon which incorporates the use of real-time PCR.
The present invention relates to a method for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), which comprises the following steps:
a) extracting deoxyribonucleic acid (DNA) from coho salmon either from biological or solid or semisolid matrices, such as muscle, fin, gill or the like, or fluids, such as total blood, plasma and serum;
optionally storing the DNA eluate obtained in step (a) at 4 C;
b) amplifying the DNA eluate from step (b) by the use of polymerase chain reaction (PCR), in real-time format, using the fluorescent probe pCsex 1 which corresponds to the sequence (FAM) 5 "-CCTGTGTATACTTGGGAGCGGTAAGCC-3" and a mixture of primers comprising the CSex2s primer: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and the CSex2r primer: 5 '-GGAACAGATGGAGCGGTCTTC-3 ';
c) identifying the presence of the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTGA
ATTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTA
TACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCT
CCATCTGTTCC, as a fluorescence signal, in the amplification resulting from step (c), assigning to the presence of said fluorescence signal the determination of male sex in coho salmon, whose DNA was amplified, and to the absence of said fluorescence signal, the determination of female sex in coho salmon, whose DNA was amplified.
The present invention also relates to a mixture of primers for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), which comprises the synthetic sequences CSex2s: 5 .-CATTATATTTGGCTGAAGAGTCAGTG-3 and CSex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 In addition, the invention comprises a kit for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), which comprises:
a) a mixture of primers comprising the synthetic sequences CSex2s: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and C Sex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 ;
b) a fluorescent probe comprising the sequence:
pCsex 1 (FAM) 5 '-CCTGTGTATACTTGGGAGCGGTAAGCC-3 "(BHQ).
Brief Description of the Figures Figure 1: Amplification curve by real-time PCR in sex determination of coho salmon, the curve represents the presence of genetic sequence only in males.
Example, methodology The extraction of genetic material can be performed from multiple sources. In the case of using solid or semisolid biological matrices, such as muscle, fin, gill, etc., the sample is placed inside a bag and is macerated with a rubber hammer. Subsequently, phosphate buffered saline (PBS) buffer is added enough to reach a suitable turbidity (equivalent to No. 5 of the McFarland scale).
Samples corresponding to fluids (total blood, plasma and serum) are directly treated.
Subsequently, the DNA (deoxyribonucleic acid) extraction is performed, for example, with a ' = CA 02821014 2013-06-10 commercial kit. For this example, the protocol of the commercial kit QIAampODNA Blood Mini Kit will be used (Qiagen; catalog No. 51104X), wherein the description of the methodology will mention solutions whose abbreviations correspond to the original ones disclosed in the commercial kit (for example, AL buffer). To begin with the DNA extraction, 200 ml of sample (homogenate or fluid, whichever the case may be) are taken and introduced into an Eppendorf tube which has not been previously used, 20 ul Proteinase K (Invitrogen) are added and it is gently homogenized to be incubated during 30 minutes at 60 C. Subsequently, 200 ul of AL
buffer are added to the sample in the tube. Homogenize by vollexing for 15 seconds and incubate at 56 C for 12 min. Briefly centrifuge at low revolution speed in order to avoid leaving drops (3000 rpm for 10 sec) on the edges. Add 200 ul of absolute ethanol (96-100%) and homogenize by vortexing for 15 seconds. Carefully add the homogenate to a column and centrifuge at 10000 x g for 1 min. Subsequently, change the collection tube to another collection tube which has not been previously used. Add 500 ul of AW1 buffer, incubate for 1 min in the column and centrifuge at 10000 x g for 1 min. Change the collection tube to another collection tube which has not been previously used. Add 500 ul of AW2 buffer, incubate for 1 min in the column and centrifuge at maximum speed for 3 min. Place the column in another collection tube which has not been previously used and add 50 ul of molecular biology grade water, and incubate at room temperature for 5 min. Finally, centrifuge at maximum speed for 1 min and store the eluate (DNA; deoxyribonucleic acid) at 4 C before use thereof.
= = CA 02821014 2013-06-10 , In order to carry out the amplification phase of the genetic material, PCR in real-time format is used. For this part of the assay, for example, a commercial kit (Express qPCR
Supermix Universal Kit (Invitrogen)) is used; for this purpose, a number of reactions based on the number of samples to be analyzed will be prepared. For example, for preparing 10 amplification reactions, prepare the microplates corresponding to this number of reactions and prepare the master mix as described in the kit; the following table shows the volumes of each component for preparing the master mix:
Master Mix 10X (u1) Express qPCR SuperMix with Premixed ROX 10 100 Fluorescent probe pCsexl (1)(O.3 uM) 10 Mixture of primers (2) (0.9 uM) 10 Nuclease-free water 70 (I) pCsex 1 (FAM, 5'-CCTGTGTATACTTGGGAGCGGTAAGCC-3' (BHQ).
FAM: 6-carboxy-fluorescein (fluorophore) BHQ: Black Hole Quencher (Quencher; fluorescent silencer) =
Nuclease-free water: water that is free of nucleases which are enzymes that degrade genetic material.
(2) Primer sequences:
C Sex2s: 5'-CATTATATTTGGCTGAAGAGTCAGTG-3' and CSex2r: 5'-GGAACAGATGGAGCGGTCTTC-3'.
Subsequently, 19 ul of the master mix are placed in each of the microtubes and 1 ul of the previously extracted DNA sample is introduced into each one. Subsequently, place the tubes in a real-time thermocycler with the following thermal profile or schedule:
1 cycle: 95 C, 20 seconds 40 cycles: 95 C, 10 seconds 55 C, 15 sec*, fluorescence reading 1 cycle: 25 C, 30 seconds At the end of the amplification schedule, the results are interpreted as detailed below.
In each microtube in which a real-time PCR reaction was conducted for discerning sex in a coho salmon sample, two events may occur. First, that a fluorescence signal is generated, which is interpreted as the presence of the Cohoseq sequence, which in turn is interpreted as male sex of the salmonid since the female should not have this region. Second, that a fluorescence signal is not generated, which is interpreted as female sex since the Cohoseq sequence is not found, and therefore, there is no substrate for the generation of a signal. Figure 1 shows an analysis of some samples, it can be observed that there are several amplification events. The amplifying curves correspond to samples that were identified as male coho salmon, while the samples that did not amplify correspond to samples that were identified as female coho salmon.
In parallel, 60 coho salmon samples were analyzed, 30 female and 30 male, fish whose sex was previously established by visual inspection since they were adult specimens.
Then, the samples were submitted to a real-time PCR analysis, obtaining 100% agreement of the results obtained with the prior information related to the sex of the samples, which was previously established by visual inspection.
Bibliography BruneIli J.P., Wertzler K.W., Sundin K and Thorgaard G.H. 2008. Y-specific sequences and polymorphisms in rainbow trout and Chinook salmon. Genome 51, 739-748.
Felip A, Fujiwara A., Young W.P., Wheeler PA, Noakes M., Phillips R.B and Thorgaard G.H.
2004. Polymorphism and differentiation of rainbow trout Y chromosomes. Genome 47, 1105-1113 .
Iturra P., Lam N., De la Fuente M., Vergara N and Medrano J.F. 2001.
Characterization of sex chromosomes in rainbow trout and coho salmon using fluorescence in situ hybridization (FISH).
Genetica 111, 125-131.
Claims (3)
1 11.
A method for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch) CHARACTERIZED in that it comprises the following steps:
a) extracting deoxyribonucleic acid (DNA) from coho salmon either from biological or solid or semisolid matrices, such as muscle, fin, gill or the like, or fluids, such as total blood, plasma and serum;
b) amplifying the DNA eluate from step (b) by the use of polymerase chain reaction (PCR), in real-time format, using the fluorescent probe pCsex 1 which corresponds to the sequence (FAM) 5"-CCTGTGTATACTTGGGAGCGGTAAGCC-3 ' and a mixture of primers comprising the CSex2s primer: 5"-CATTATATTTGGCTGAAGAGTCAGTG-3' and the CSex2r primer: 5'-.
GGAACAGATGGAGC GGTCTTC-3 ' ;
c) identifying the presence of the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTGA
ATTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTA
TACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCT
CCATCTGTTCC, as a fluorescence signal, in the amplification resulting from step (c), assigning to the presence of said fluorescence signal the determination of male sex in coho salmon, whose DNA was amplified, and to the absence of said fluorescence signal, the determination of female sex in coho salmon, whose DNA was amplified.
A method for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch) CHARACTERIZED in that it comprises the following steps:
a) extracting deoxyribonucleic acid (DNA) from coho salmon either from biological or solid or semisolid matrices, such as muscle, fin, gill or the like, or fluids, such as total blood, plasma and serum;
b) amplifying the DNA eluate from step (b) by the use of polymerase chain reaction (PCR), in real-time format, using the fluorescent probe pCsex 1 which corresponds to the sequence (FAM) 5"-CCTGTGTATACTTGGGAGCGGTAAGCC-3 ' and a mixture of primers comprising the CSex2s primer: 5"-CATTATATTTGGCTGAAGAGTCAGTG-3' and the CSex2r primer: 5'-.
GGAACAGATGGAGC GGTCTTC-3 ' ;
c) identifying the presence of the sequence CATTATATTTGGCTGAAGAGTCAGTGTTCTGCTTGTATTAATATAATATAGAGCTTGA
ATTTAGACTTGAATTCTCACAGTTGGAGGTCAGTTTAGTTATGAATTAAACCTGTGTA
TACTTGGGAGCGGTAAGCCATGTGCCCATTTACAAAGTCTGACCAGAGAAGACCGCT
CCATCTGTTCC, as a fluorescence signal, in the amplification resulting from step (c), assigning to the presence of said fluorescence signal the determination of male sex in coho salmon, whose DNA was amplified, and to the absence of said fluorescence signal, the determination of female sex in coho salmon, whose DNA was amplified.
2. Mixture of primers for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), CHARACTERIZED in that it comprises the synthetic sequences CS ex2s: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and C Sex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 '.
3. Kit for discerning sex through the use of real-time polymerase chain reaction (PCR) in coho salmon (Oncorhynchus kisutch), CHARACTERIZED in that it comprises:
a) a mixture of primers comprising the synthetic sequences C Sex2s: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and CSex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 ';
b) a fluorescent probe comprising the sequence:
pCsex 1 (FAM) 5 '-CCTGTGTATACTTGGGAGCGGTAAGCC-3 '(BHQ).
a) a mixture of primers comprising the synthetic sequences C Sex2s: 5 '-CATTATATTTGGCTGAAGAGTCAGTG-3 ' and CSex2r: 5 '-GGAACAGATGGAGCGGTCTTC-3 ';
b) a fluorescent probe comprising the sequence:
pCsex 1 (FAM) 5 '-CCTGTGTATACTTGGGAGCGGTAAGCC-3 '(BHQ).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CL1418-2010 | 2010-12-10 | ||
| CL2010001418A CL2010001418A1 (en) | 2010-12-10 | 2010-12-10 | Method to detect sex by using the human polymerase chain reaction (pcr) in real time in salmon coho. |
| PCT/CL2011/000076 WO2012075599A1 (en) | 2010-12-10 | 2011-12-09 | Sex discernment by real-time pcr in coho salmon |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2821014A1 true CA2821014A1 (en) | 2012-06-14 |
Family
ID=46206505
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2821014A Abandoned CA2821014A1 (en) | 2010-12-10 | 2011-12-09 | Sex discernment by real-time pcr in coho salmon |
Country Status (7)
| Country | Link |
|---|---|
| AU (1) | AU2011340101A1 (en) |
| CA (1) | CA2821014A1 (en) |
| CL (1) | CL2010001418A1 (en) |
| GB (1) | GB2500155A (en) |
| NO (1) | NO20130822A1 (en) |
| NZ (1) | NZ612470A (en) |
| WO (1) | WO2012075599A1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5480774A (en) * | 1993-10-14 | 1996-01-02 | A/F Protein, Inc. | Determination of genomic sex in salmonids |
-
2010
- 2010-12-10 CL CL2010001418A patent/CL2010001418A1/en unknown
-
2011
- 2011-12-09 NZ NZ612470A patent/NZ612470A/en not_active IP Right Cessation
- 2011-12-09 GB GB1311625.6A patent/GB2500155A/en not_active Withdrawn
- 2011-12-09 AU AU2011340101A patent/AU2011340101A1/en not_active Abandoned
- 2011-12-09 CA CA2821014A patent/CA2821014A1/en not_active Abandoned
- 2011-12-09 WO PCT/CL2011/000076 patent/WO2012075599A1/en active Application Filing
-
2013
- 2013-06-12 NO NO20130822A patent/NO20130822A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| NO20130822A1 (en) | 2013-06-28 |
| GB201311625D0 (en) | 2013-08-14 |
| WO2012075599A1 (en) | 2012-06-14 |
| CL2010001418A1 (en) | 2011-04-25 |
| GB2500155A (en) | 2013-09-11 |
| AU2011340101A1 (en) | 2013-07-11 |
| NZ612470A (en) | 2013-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10981137B2 (en) | Enrichment of DNA sequencing libraries from samples containing small amounts of target DNA | |
| JP6335918B2 (en) | Target enrichment without restriction enzymes | |
| CA3076518A1 (en) | Crispr effector system based diagnostics | |
| EP3087202B1 (en) | Nucleic acid detection method and kit | |
| US9290815B2 (en) | Method for quantifying human DNA | |
| JP2015521468A (en) | Compositions and methods for negative selection of unwanted nucleic acid sequences | |
| WO2010083250A2 (en) | Single-cell nucleic acid analysis | |
| CA2905410A1 (en) | Systems and methods for detection of genomic copy number changes | |
| AU2012236880A1 (en) | Telomere length measurement in formalin-fixed, paraffin embedded (FFPE) samples by quantitative PCR | |
| JP2015516814A (en) | Enrichment and sequencing of targeted DNA | |
| CA2688571A1 (en) | Methods of directly amplifying nucleic acids from crude samples | |
| Mitsunaga et al. | Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity | |
| CN108220450B (en) | Identification method and identification kit for animal-derived components in meat products | |
| CN106609299B (en) | Kit for determining concentration of fetal free DNA in plasma of pregnant woman | |
| CN107988334B (en) | Method for SNP typing by direct PCR of oral swab | |
| US20150353918A1 (en) | Method and materials for nucleic acids extraction and purification | |
| CA2821014A1 (en) | Sex discernment by real-time pcr in coho salmon | |
| CA3041287A1 (en) | Chromosomal assessment by rapid lamp analysis | |
| Sun et al. | Analysis of mitochondrial DNA copy number and its regulation through DNA methylation of POLGA | |
| WO2015042649A1 (en) | A quantitative assay for target dna in a mixed sample comprising target and non-target dna | |
| JP6853523B2 (en) | PCR using helicase | |
| Neduvat et al. | Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA’s for HLA typing | |
| Thanakiatkrai et al. | An investigation into the protective capabilities of nucleosomes on forensic STRs | |
| JP2023103372A (en) | Improved nucleic acid target enrichment and related methods |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |
Effective date: 20171211 |