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WO2016008884A1 - Procédé permettant de détecter une amplification par pcr dans un échantillon - Google Patents

Procédé permettant de détecter une amplification par pcr dans un échantillon Download PDF

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WO2016008884A1
WO2016008884A1 PCT/EP2015/066063 EP2015066063W WO2016008884A1 WO 2016008884 A1 WO2016008884 A1 WO 2016008884A1 EP 2015066063 W EP2015066063 W EP 2015066063W WO 2016008884 A1 WO2016008884 A1 WO 2016008884A1
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sequence
tail
label
pcr primer
allele
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Janika Higgins
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Identigen Ltd
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Identigen Ltd
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Priority claimed from EP14176969.5A external-priority patent/EP2975136A1/fr
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/319—Exonuclease
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/101—Interaction between at least two labels
    • C12Q2565/1015—Interaction between at least two labels labels being on the same oligonucleotide

Definitions

  • the invention relates to a method of detecting PCR amplification in a sample.
  • the invention relates to a method of genotyping a sample of DNA for the presence of a first or second allele of a locus within the DNA by means of PCR amplification.
  • genotyping assay systems there are several genotyping assay systems available: one example disclosed in WO02/34947 is a PCR based method for genotyping SNP's with high sensitivity that employs a primer pair, in which the forward primer has a tail that does not hybridise to the target sequence, and a dual labelled probe.
  • the probe comprises a reported label, a quencher, and a sequence identical to the tail of the forward primer. Detection by the probe occurs after the third PCR round.
  • KASP chemistry EP 1726664. This detection system is based on two singly labelled (probe) oligonucleotides, which are hybridised together when the target sequence for the reporter oligonucleotide is not present.
  • One of the oligonucleotides is labelled with a reporter dye and the other with a quencher molecule, thus, the fluoresence of the reporter is quenched (undetectable) when the probe is in its hybridised form, commonly known as a probe cassette.
  • a probe cassette commonly known as a probe cassette.
  • the KASP detection system also utilises two allele specific and a single shared common primers which are designed to target the SNP site. These two allele specific primers are "tailed" with either one of two sequences which are complementary to one of the two universal probes.
  • the probe When the allele specific primer is used in the PCR reaction, the probe will hybridise with the "tail" sequence now present in the newly synthesised DNA, releasing reporter dye from the proximity of the quencher molecule resulting in a fluoresence signal.
  • KASP chemistry is known to have technical constraints with “over-cycling” leading to the changing of fluorescence over time due to random amplification, inappropriate binding, etc. This leads to issues when trying to genotype multiple samples with varying template DNA quality and concentration, as not all samples reach a fluorescence plateau at the same cycle number. This means that each sample for each probe mix must be analysed multiple times to "track" the fluorescence to best estimate which probe is amplified the most for each sample.
  • KASP probes can become randomly annealed to other points along the sequence as the supplies within the reagent mix are depleted, leaving the fluorophores from either cassette to be cleaved or separated from the quencher molecule and fluoresce permanently regardless of whether or not they should have taken part in the PCR reaction. This is why we see a movement of the clusters either towards the heterozygous group or into a scatter gun style pattern that characterises the issues with KASP performance.
  • the invention provides a method for detecting the presence of a target DNA molecule in a sample. Similar to the KASP methodology, the methods of the invention employ an unlabelled tailed forward primer and a common reverse primer that are used to initiate synthesis of a target DNA molecule. However, instead of employing a probe that consists of labelled first and second oligonucleotide sequences that hybridise to one another in free solution, the method of the invention employs a dual labelled single stranded probe. Surprisingly, the use of dual labelled probes has been shown to reduce the problem of "over-cycling" to an insignificant number of mis-calls (in comparison to KASP data)/change of calls being reported over multiple cycling. Therefore only one read is necessary, leading to dramatically reduced data review and higher throughput of data compared to its KASP equivalent.
  • the method of the invention is referred to as IDENTISNPTM
  • the invention provides a method for detecting PCR amplification of a target DNA molecule in a sample, which method employs:
  • a forward PCR primer having a sequence that is complementary to the target double stranded nucleic acid and a tail sequence
  • oligonucleotide probe binds to the sequence complementary to the tail region, whereby the oligonucleotide probe is displaced and cleaved resulting in a detectable signal from the reporter label.
  • the invention also provides a kit suitable for performing a method of detecting PCR amplification of a target double stranded nucleic acid in a sample, the kit comprising
  • a forward PCR primer having a sequence that is complementary to the target double stranded nucleic acid and a tail sequence
  • a dual labelled probe containing an oligonucleotide sequence identical to the tail sequence of the forward PCR primer oligonucleotide, and a reporter label and a quencher-label separated by a nuclease susceptible cleavage site.
  • the invention provides a method of genotyping a sample of DNA for the presence of a first or second allele of a locus within the DNA by means of PCR amplification, which method employs:
  • a first labelled probe comprising an oligonucleotide sequence identical to the tail sequence of the first forward PCR primer, and a first reporter label and a quencher-label separated by a nuclease susceptible cleavage site;
  • a second labelled probe containing an oligonucleotide sequence identical to the tail sequence of the second forward PCR primer, and a second reporter label and a quencher-label separated by a nuclease susceptible cleavage site, in which the second reporter label emits a signal that is different to that of the first reporter label
  • the invention provides a method of genotyping a sample of DNA for the presence or absence of a single nucleotide polymorphism (SNP) within the DNA by means of PCR amplification, which method employs:
  • a first labelled probe comprising an oligonucleotide sequence identical to the tail sequence of the first forward PCR primer, and a first reporter label and a quencher-label separated by a nuclease susceptible cleavage site;
  • a second labelled probe containing an oligonucleotide sequence identical to the tail sequence of the second forward PCR primer, and a second reporter label and a quencher-label separated by a nuclease susceptible cleavage site, in which the second reporter label emits a signal that is different to that of the first reporter label
  • the invention also provides a kit suitable for genotyping a sample of DNA for the presence of a first or second allele of a locus within the DNA (or the presence or absence of a SNP in a sample of DNA), the kit comprising:
  • first forward PCR primer having a first allele-specific sequence and a first tail sequence
  • second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence
  • a first labelled probe comprising an oligonucleotide sequence identical to the tail sequence of the first forward PCR primer, and a first fluorescent label and a quencher-label separated by a nuclease susceptible cleavage site;
  • a second labelled probe containing an oligonucleotide sequence identical to the tail sequence of the second forward PCR primer, and a second fluorescent label and a quencher-label separated by a nuclease susceptible cleavage site, in which the second fluorescent has excitation and emission properties that are different to those of the first fluorescent label.
  • PCR should be understood to mean polymerase chain reaction, which is fully described in US Patent No: 4683195 (the contents of which are incorporated herein by reference).
  • Performing PCR on a sample generally involves use of Taq polymerase, dNTP' s and reaction buffer.
  • the kit comprises Taq polymerase, dNTP's and reaction buffer.
  • the reaction buffer is selected from (but not limited to) the following list:
  • detecting PCR amplification of a target DNA molecule in a sample should be understood to mean determining whether a target DNA molecule is present in a sample, or quantitatively determining the presence of the target DNA in the sample.
  • forward PCR primer should be understood to mean a short single stranded oligonucleotide sequence designed to act as a point of initiation of synthesis along a complementary part of a first strand of a target DNA molecule.
  • reverse primer or common reverse primer
  • tail sequence refers to a part of the forward PCR primer, typically at the 5' end of the primer molecule that is not complementary to the target DNA molecule. Typically, the tail sequence is 20-30, preferably 20-24, nucleotides in length.
  • the term "dual labelled probe” refers to a single stranded oligonucleotide sequence having a reporter label, a quencher label, and probe sequence disposed between the two labels, and in which the reporter label and quencher label are in sufficient proximity such that the quencher prevents the reporter label emitting a detectable signal.
  • the probe sequence generally includes a sequence that is sensitive to a Taq polymerase. Typically, the probe comprises 20-30 nucleotides, preferably 20-24 nucleotides. In one embodiment, the dual labelled probe is selected from the sequences:
  • reporter label should be understood to mean a molecule that is capable of emitting a detectable signal and is capable of being quenched by the quencher molecule.
  • reporter molecules include molecules that are detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • reporter labels that may be employed include enzymes, enzyme substrates, radioactive atoms, fluorescent dyes, chromophores, chemiluminescent labels, or ligands having specific binding partners.
  • the reporter label is detectable by spectroscopy, and ideally is a fluorescent dye.
  • fluorescent dyes examples include FAM, VIC, LIZ, NED, PET.
  • first and second fluorescent reporter labels include FAM (abs 495nm, em520nm) and JOE (abs 520nm, em 548nm).
  • quencher label should be understood to mean a molecular entity that is capable of quenching the signal emitted by the reporter molecule, when in proximity with the reporter molecule.
  • the quencher is typically selected from BHQ-1, BHQ-2, BHQ-3, TAMRA.
  • Taq polymerase should be understood to mean a DNA polymerase that has a DNA synthesis dependent, strand replacement 5 '-3' exonuclease activity (Gelfand, PCR Technology: Principles and Applications for DNA Amplification, Erlich, Ed., Stockton Press, N.Y. (1989), Chapter 2).
  • the term "genotyping a sample of DNA to detect first or second alleles of a gene within the DNA sample” should be understood to mean determining whether a given allele of a gene or locus is present in the sample of DNA.
  • the method of the invention can be employed to detect the presence or absence of single nucleotide polymorphisms in a sample, by using the method of the invention to detect an allele comprising the SNP.
  • the reporter molecules on the two probes will be different, thus allowing the signal emitted during the PCR process to be assigned to one or the other probe.
  • allele-specific primer as applied to the forward primers of the invention should be understood to mean that the 3' terminal base of the primer is complementary to the allele - specific polymorphic base at the SNP site.
  • specific to the first allele as applied to the forward primers of the invention should be understood to mean that the 3' terminal base of the primer is complementary to the first allele- specific polymorphic base at the SNP site.
  • sample should be understood to mean a sample comprising or consisting of DNA.
  • the sample is a biological sample, for example a biological fluid selected from blood or a blood derived product such as plasma or serum, saliva, urine, sweat, or cerebrospinal fluid.
  • the sample may also be a sample of cells or tissue, for example muscle, nail, hair, bone, marrow, brain, vascular tissue, kidney, liver, peripheral nerve tissue, skin, epithelial tissue.
  • the tissue or cells may be normal or pathological tissue.
  • the sample will be treated to remove all material except DNA.
  • PCR sample preparation protocols are well described in the literature and are available from the websites of Agilent, Life Technologies, Qiagen and Illumina.
  • the sample comprises DNA of unknown origin and the method of the invention may be employed to detect the presence (or quantify the amount, if present) of a target DNA in the sample.
  • the method of the invention may be employed to determine the presence of a particular allele of a DNA locus, for example the presence of a SNP in a gene or other locus in DNA.
  • IDENTISNPTM chemistry can be used to identify the presence of known SNPs within a DNA sample. With a panel of SNPs that have been determined through investigation to provide enough unique identifying information so as to be used to both give a unique ID for the sample tested as well as using Mendelian inheritance, used to determine the parents of a sample provided.
  • Figs 1A to 1J is an illustration of the steps of a method of detecting PCR amplification of a target nucleic acid according to the invention
  • Figs. 2A to 2H is an illustration of the steps of a method of genotyping a sample of DNA for the presence of a first or second allele of a locus within the DNA by means of PCR amplification.
  • a method of detecting PCR amplification of a target sequence there is illustrated a method of detecting PCR amplification of a target sequence according to the invention.
  • a target DNA molecule I there is illustrated a target DNA molecule I, a forward PCR primer 2 having a first sequence 3 complementary to a sequence at the 5' end of the first strand of the target DNA molecule I, a reverse primer 4 having a sequence complementary to a sequence at the 5' end of the second strand of the target DNA molecule I, and a probe 5 having a fluorescent label 6, a quencher 7, and a probe sequence 8.
  • the forward PCR primer 2 additionally comprises a tail sequence 10 that is identical to the probe sequence 8.
  • a first round of PCR is initiated with a denaturation step and the DNA molecule I denaturing to form single stranded DNA molecules II and III.
  • the forward PCR primer 2 binds to the 5' end of strand II
  • the reverse PCR primer 4 binds to the 5' end of the second strand III, in an annealing step.
  • the probe sequence 8 of the probe 5 is identical with the tail sequence 10 of the primer 2, it will not bind to the tail.
  • FIG. 1C there is illustrated a synthesised DNA molecule IV formed as a result of the first round of PCR and elongation of strand II of Fig. IB, the DNA molecule incorporating the tail sequence 10.
  • the probe sequence 8 of the probe 5 is identical with the tail sequence 10 of the primer 2, it will not bind to the tail.
  • a second round of PCR is initiated with a denaturation step and the DNA molecule IV denaturing to form single stranded DNA molecules V and VI.
  • the forward PCR primer 2 binds to the 5' end of strand V
  • the reverse PCR primer 4 binds to the 5' end of the second strand VI, in an annealing step.
  • the probe sequence 8 of the probe 5 is identical with the tail sequence 10 of the primer 2, it will not bind to the tail.
  • a third round of PCR is initiated with a denaturation step and the DNA molecule VIII denaturing to form single stranded DNA molecules IX and X.
  • Single stranded DNA molecule IX comprises a tail region 12 that is complementary to the tail region 10, and therefore complementary to the probe sequence 8.
  • the probe 5 will therefore bind to the tail region 12 of molecule IX.
  • the 5'- 3'exonuclease activity of a Tag polymerase will cleave the dual labelled probe, releasing the quencher label 7 into solution and allowing the fluorescent label to emit a detectable fluorescent signal.
  • Fig. 1H shows a newly synthesised DNA molecule incorporating the fluorescent label 6.
  • a fourth round of PCR is initiated with a denaturation step and the DNA molecule of Fig. 1H denaturing to form two single stranded DNA molecules, one comprising a tail region 12 that is complementary to the tail region 10, and therefore complementary to the probe sequence 8.
  • the probe 5 therefore binds to the tail region and during the elongation step, the 5'-3'exonuclease activity of a Tag polymerase will cleave the dual labelled probe, again releasing the quencher label 7 into solution and allowing the fluorescent label to emit a detectable fluorescent signal.
  • Fig. 1J shows a newly synthesised DNA molecules incorporating the fluorescent label 6 and providing continued amplification of signal and no loss of data/signal quality.
  • This embodiment of the invention may be employed to detect the presence of a specific target DNA molecule within a sample, by designing primer and probes that are specific for PCR amplification of the target sequence.
  • the method of the invention may be employed to detect a specific allele of the target DNA molecule, for example an allele comprising a single nucleotide polymorphism, in which the reaction mixture comprises a forward PCR primer specific for a first allele of the target DNA and having a first tail, and a second forward PCR primer specific for a second allele of the target DNA and having a second tail, and two dual labelled probes, one having a probe sequence identical to the first tail and the other having a probe sequence identical to the second tail.
  • FIGs 2A to 2H there is illustrated a further embodiment of the invention, specifically a method of genotyping a sample of DNA for the presence of a first or second allele of a locus within the DNA by means of PCR amplification, in which parts identified with reference to Figs 1A to 1H are assigned the same reference numerals.
  • a first allele of a target DNA molecule I incorporating a single polynucleotide polymorphism (SNP) X
  • a first forward PCR primer 20A specific for the first allele of the target DNA molecule and having a first sequence 30A complementary to a sequence at the 5' end of the first strand of the target DNA molecule I and including a 3' terminal base that is complementary with the polymorphic base X in the first allele of the target DNA molecule I
  • a second forward PCR primer 20B specific for the second allele of the target DNA molecule and having a first sequence 3 OB complementary to a sequence at the 5' end of the first strand of the target DNA molecule I and including a 3' terminal base that is complementary with the polymorphic base (not shown) in the second allele of the target DNA molecule I
  • a common reverse primer 40 having a sequence complementary to a sequence at the 5' end of the second strand of the target DNA molecule I
  • a first probe having a sequence complementary to a
  • the first forward PCR primer 20A additionally comprises a tail sequence 100 A that is identical to the first probe sequence 80A.
  • the second forward PCR primer 20B additionally comprises a tail sequence 100B that is identical to the second probe sequence 80B.
  • the figures do not illustrate the other components required to perform PCR, which include Taq DNA polymerase, deoxy nucleotide triphosphates dNTP's, and reaction buffer, the details of which will be known to a person skilled in the art.
  • a first round of PCR is initiated in the same way as described with reference to Figs. IB and 1C.
  • the first forward PCR primer 20A which is specific for the first allele of the target DNA molecule anneals to the first strand II and the common reverse primer anneals to the second strand III, and following elongation two new double stranded DNA molecules IV and V are formed, with molecule IV including the tail sequence 100 A (Fig. 2C).
  • a second round of PCR is initiated with a denaturation step and the DNA molecule IV denaturing to form single stranded DNA molecules VI and VII.
  • the first forward PCR primer 20 A binds to the 5' end of strand VI
  • the common reverse PCR primer 40 binds to the 5' end of the second strand VI, in an annealing step.
  • the probe sequence 80A of the probe 50A is identical with the tail sequence 100A of the primer 20A, it will not bind to the tail. Referring to Fig.
  • the probe 50A will therefore bind to the tail region 120A of molecule XL During the elongation step, the 5 '-3 'exonuclease activity of a Tag polymerase will cleave the dual labelled probe, releasing the quencher label into solution and allowing the fluorescent label to emit a detectable fluorescent signal.
  • samples that were previously tested and assigned SNP genotypes using KASP chemistry were selected to be re tested with the IDENTISNPTM chemistry. From these samples and KASP results, we were able to designate parentage trios (one male, one female, one offspring). We have selected 80 female parents, 80 male parents and 220 offspring. These samples had already been prepared for the KASP chemistry and the same samples were then retyped using IDENTISNPTM chemistry. In order to assess the success of the IDENTISNPTM chemistry in using one call, we compared both the first call and a typical recycling program identical to KASPs recommended re-cycling procedure.
  • the method of the invention was performed according to the PCR cycling conditions shown in Table 1 and employing the procedure described in detail (in the "detailed description of the invention” section, referring specifically to figures 2A - 2H).
  • the template DNA for each sample was run against a panel of SNP targets designed for Salmon parentage analysis, 65 in total. Each test for the presence of an allele consisted of particulars as described in section "Brief Description of the Invention" paragraph 5.
  • the primers, probes and template DNA were incubated together in a PCR mastermix (consisting of all the components necessary to perform PCR, with the exception of template, primers, and probes). Following on from this the PCR protocol was initiated in line with the cycling conditions as described in Table 1. Cycling Conditions
  • SNP concordance rate with KASP is >99% in both the single read and multiple read analysis using the IDENTISNPTM chemistry (see Table 3.).
  • Fin clips were taken from a set of 3 specific salmon groups that were designated as female parents, male parents and offspring.
  • the aim of the experiment was to identify the family trio of male and female parent and their offspring using SNP genotyping and a panel of 65 markers previously designed and selected for salmon parentage. This would then be compared against a previous experiment where similar mating structures were observed and KASP chemistry was used.
  • the fin clips were lysed in a proteinase K buffer overnight. These samples were then cleaned using a magnetic bead based DNA isolation kit (eg Mag mini DNA extraction kit). After the samples were cleaned, the template DNA for each sample was run against the panel of SNPs, each test for the presence of an allele consisted of particulars as described in paragraph 1, page 4, (detailed more in depth in the Example of IDENTISNPTM chemistry in practice section, page 6). The primers, probes (as described in the "brief description of the invention", page 5) and template DNA were incubated together in a PCR mastermix (consisting of all the components necessary to perform PCR, with the exception of template, primers, and probes). Following on from this the PCR protocol was initiated in line with the cycling conditions as described in Table 1.
  • a PCR mastermix consisting of all the components necessary to perform PCR, with the exception of template, primers, and probes.
  • Scoring time was also analysed among the lab analysts, comparing the time necessary to score the KASP multiple reads (3 in this case) to the amount of time needed to score the IDENTISNPTM single read. On average there has been a reduction in scoring time of circa 75%, with times varying between analysts and data sets.
  • the IDENTISNPTM chemistry single read is at least the equivalent of the KASP chemistry re cycling protocol and has numerous benefits including most importantly reduced scoring time and a reduction in error rates (i.e. miscalling of SNPs) with a >8 fold reduction in per locus Mendelian error rate and the same in per offspring Mendelian error rate (Table 7. vs. Table 8.).
  • Example 3
  • Samples were selected with an aim to minimise potential external factors having an effect on the sample quality or genotyping. To this end we have selected 3 sample types from 3 different customers, with data from the 1 st January 2014 until the 31 st of March 2014 provided by KASP, and data from the 1 st January 2015 until the 31 st of March 2015 provided by IDENTISNPTM. Samples were prepared in the same way over both time periods with no change in the sample preparation protocol. Sample collection method has not changed over this time period either for any of the three customers. Genotype scoring and data analysis was done by the same team over both time periods, and none had been notified that this data may be used for any other purpose than for customers.
  • KASP was run in line with their own recommended protocol, detailed below in Table 11, while IDENTISNPTM was run with the protocol detailed in Table 12.
  • the large number of data points over the given time periods provides a suitable volume of data for comparison between the chemistry types.
  • the sample collection, lab handling and data analysis was thoroughly investigated to ensure that there had been no change to personnel or protocol that could be seen to change overall call rates outside of the change in chemistries themselves. These samples were tested without the knowledge that they would be used in a data analysis comparison between the two chemistries, therefore removing any potential bias by the investigator or other staff members.
  • Tables 9 and 10 detail the comparable PCR protocols for IDENTISNPTM and KASP, when the direct PCR time is calculated (3840 seconds for KASP, 2980 seconds for IDENTISNPTM), we see an immediate time saving of 22.39% in direct PCR time. While there will be additional time for both chemistries dependent on what machinery is used, the time saving in seconds will not change (860 seconds). In high throughput labs this is equivalent to one extra PCR when using IDENTISNPTM for every 5 PCRs run with KASP (machinery dependent). This does not take into account the "recycling" protocol used by KASP where by the "amplification” step in their protocol (Table 9.) is re-run twice for 3 cycles each, to track the movement of their genotype data.

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Abstract

Cette invention concerne un procédé de détection d'une amplification par PCR d'une molécule d'ADN cible dans un échantillon qui utilise une amorce de PCR sens ayant une séquence qui est complémentaire de l'acide nucléique double brin cible et une séquence de queue, une amorce de PCR inverse ayant une séquence qui est complémentaire de l'acide nucléique double brin cible, et une double sonde marquée contenant une séquence oligonucléotidique identique à la séquence de queue de l'oligonucléotide amorce de PCR sens, et une étiquette rapporteur et une étiquette extincteur séparées par un site de clivage sensible à une nucléase. Le procédé comprend les étapes consistant à incuber les amorces de PCR sens et inverse et la double sonde marquée ensemble, à mettre en œuvre au moins deux tours de PCR pour générer un acide nucléique double brin comprenant la région de queue et une séquence complémentaire de la région de queue, et à lancer un autre tour de PCR pour que la sonde oligonucléotidique se lie à la séquence complémentaire de la région de queue, de façon que la sonde oligonucléotidique soit déplacée et clivée avec pour effet la génération d'un signal détectable émanant de l'étiquette rapporteur.
PCT/EP2015/066063 2014-07-14 2015-07-14 Procédé permettant de détecter une amplification par pcr dans un échantillon Ceased WO2016008884A1 (fr)

Applications Claiming Priority (4)

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EP14176969.5 2014-07-14
EP14176969.5A EP2975136A1 (fr) 2014-07-14 2014-07-14 Procédé de détection de l'amplification par PCR dans un échantillon
EP14188259 2014-10-09
EP14188259.7 2014-10-09

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WO2016008884A1 true WO2016008884A1 (fr) 2016-01-21

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CN112969707A (zh) * 2018-09-07 2021-06-15 克罗玛科德公司 用于单核苷酸多态性的多重检测的具有多个结合基序的通用尾引物

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US12454720B2 (en) 2018-04-17 2025-10-28 ChromaCode, Inc. Methods and systems for multiplex analysis
US12203129B2 (en) 2018-07-03 2025-01-21 ChromaCode, Inc. Formulations and signal encoding and decoding methods for massively multiplexed biochemical assays
GB2587178B (en) * 2019-01-09 2021-10-06 3Cr Bioscience Ltd Method
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CN108774639A (zh) * 2018-05-31 2018-11-09 澳門帝傑數碼基因有限公司 一种定向聚合的荧光探针pcr
CN108774639B (zh) * 2018-05-31 2023-05-30 澳門帝傑數碼基因有限公司 一种定向聚合的荧光探针pcr
CN112969707A (zh) * 2018-09-07 2021-06-15 克罗玛科德公司 用于单核苷酸多态性的多重检测的具有多个结合基序的通用尾引物

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IES20150206A2 (en) 2016-03-09
GB201512284D0 (en) 2015-08-19

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