IES20150206A2 - A method for detecting PCR amplification in a sample - Google Patents

Abstract

A method for detecting PCR amplification of a target DNA molecule in a sample is described and employs a forward PCR primer having a sequence that is complementary to the target double stranded nucleic acid and a tail sequence, a reverse PCR primer having a sequence that is complementary to the target double stranded nucleic acid, and 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 method comprises the steps of incubating the forward and reverse PCR primers and dual labelled probe together, performing at least two rounds of PCR to generate a double stranded nucleic acid comprising the tail region and a sequence complementary to the tail region, and initiating a further round of PCR in which the 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.

Description

A method for detecting PCR amplification in a sample.

Technical Field The invention relates to a method of detecting PCR amplification in a sample. In particular, 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.

Background to the Invention 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. A further well known example of a genotyping system is KASP chemistry (EPl 726664). 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. In a bi-allelic SNP detection system, two types of these universal probes are needed. 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. 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. Over time 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.

It is an object of the invention to overcome at least one of the above-referenced problems.

Brief Description of the Invention Broadly, 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. Hereafter, the method of the invention is referred to as IDENTISNP™ Broadly, 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; - a reverse PCR primer having a sequence that is complementary to the target double stranded nucleic acid; and - 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 method comprising the steps of: - incubating the forward and reverse PCR primers, dual labelled probe, and sample DNA together; - performing at least two rounds of PCR to generate a double stranded nucleic acid comprising the tail region and a sequence complementary to the tail region; and - initiating a further round of PCR in which the 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 reverse PCR primer having a sequence that is complementary to the target double stranded nucleic acid; and - 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.

In another aspect, 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 forward PCR primer having a first allele-specific sequence and a first tail sequence; - a second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence; - a common reverse PCR primer; - 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; and - 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 method comprising the steps of: - incubating the forward and reverse PCR primers, probes, and sample DNA together; - initiating at least two rounds of PCR to generate a double stranded nucleic acid comprising the first or second tail region and a sequence complementary to the first or second tail region; - initialling a further round of PCR in which one of the probes binds to the sequence complementary to the first or second tail region, whereby the oligonucleotide probe is displaced and cleaved resulting in a detectable signal from the reporter label; and - correlating the detectable signal from the reporter label with the presence ofthe first or second allele of the locus in the sample of DNA.

In another aspect, 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 forward PCR primer having a first allele-specific sequence and a first tail sequence; - a second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence; - a common reverse PCR primer; - 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; and - 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 method comprising the steps of: - incubating the forward and reverse PCR primers, probes, and sample DNA together; - initiating at least two rounds of PCR to generate a double stranded nucleic acid comprising the first or second tail region and a sequence complementary to the first or second tail region; - initialling a further round of PCR in which one of the probes binds to the sequence complementary to the first or second tail region, whereby the oligonucleotide probe is displaced and cleaved resulting in a detectable signal from the reporter label; and - correlating the detectable signal from the reporter label with the presence ofthe first or second allele of the sample of DNA (i.e. presence or absence of the SNP).

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: - a first forward PCR primer having a first allele-specific sequence and a first tail sequence; - a second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence; - a common reverse PCR primer; - 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; and - 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.

In this specification, the term “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 details of these reagents will be known to a person skilled in the art. In one embodiment of the invention, the kit comprises Taq polymerase, dNTP’s and reaction buffer. In one embodiment, the reaction buffer is selected from (but not limited to) the following list: • TaqMan® Environmental Master Mix 2.0 (Catalog number: 4396838) • TaqMan® Genotyping Master Mix (Catalog number: 4371355) • TaqMan® GTXpress™ Master Mix (Catalog number: 4401892) • FastStart TaqMan® Probe Master (Product No. 04673409001) • Premix Ex Taq™ (Probe qPCR) (Catalog number: RR390A) • Toyobo Realtime PCR Master Mix (Catalog number: QPK-101T) In this specification, the term “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.

The term “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.

The term “reverse primer” or common reverse 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 second strand of a target DNA molecule.

The term “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: FAM-5’ GAA GGT GAC CAA GTT CAT GCT 3’- BHQ1 (SEQ ID NO: 1) JOE-5’ GAA GGT CGG AGT CAA CGG ATT 3’ - BHQ1 (SEQ ID NO: 2) The term “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. Examples of reporter molecules include molecules that are detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of reporter labels that may be employed include enzymes, enzyme substrates, radioactive atoms, fluorescent dyes, chromophores, chemiluminescent labels, or ligands having specific binding partners. Preferably, the reporter label is detectable by spectroscopy, and ideally is a fluorescent dye. Examples of fluorescent dyes that may be employed with the methods of the present invention include FAM, VIC, LIZ, NED, PET. In embodiments of the invention directed to genotyping a sample of DNA to detect the presence of a first or second allele of the DNA, two different dual-labelled probes will be employed, one having a first reporter label capable of emitting a first signal, and the second having a second reporter label capable of emitting a second signal that is different to the first signal. Examples of first and second fluorescent reporter labels include FAM (abs 495nm, em520nm) and JOE (abs 520nm, em 548nm).

The term “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. When the reporter label is a fluorescent dye, the quencher is typically selected from BHQ-1, BHQ-2, BHQ-3, TAMRA.

The term “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. Thus, 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. Generally, with these methods, it is necessary to include in the reaction mixture two forward primers, one specific to a first allele and a second specific to the second allele, and two dual labelled probes, one specific for the first forward primer and the second specific for the second primer. 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.

The term “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 allelespecific polymorphic base at the SNP site. Likewise, the term “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.

The term “sample” should be understood to mean a sample comprising or consisting of DNA. Generally, 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. Generally, 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. In one embodiment, 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. In terms of genotyping, 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.

Example of IDENTISNP™ chemistry in practice As described previously, and in more detail in figures 2A to 2H, IDENTISNP™ 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.

In this situation, all known parents are genotyped against the panel of SNPs using the IDENTISNP™ chemistry. Subsequent to this the offspring are also genotyped. Using SNP signature analysis, we can then determine which parents belong to which offspring with high probability. From this data then we can assign parents from within the group to the offspring or we can confirm that the parents of those offspring are not contained within the tested parental group.

The use of IDENTISNP™ in this way has been compared with previous similar work done in house with KASP chemistry (see Experimental examples. Example 1).

Brief Description of the Figures Figs 1A to IJ is an illustration of the steps of a method of detecting PCR amplification of a target nucleic acid according to the invention; and 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.

Detailed Description of the Invention Referring to the Figures, and initially to Figs 1A to 1H, there is illustrated a method of detecting PCR amplification of a target sequence according to the invention.

Referring initially to Fig. 1A, 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. 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. Referring to Fig. IB, 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, and the reverse PCR primer 4 binds to the 5’ end of the second strand III, in an annealing step. As 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. Referring to Fig. IC, 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. Again, as 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.

Referring to Fig. ID, 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, and the reverse PCR primer 4 binds to the 5’ end of the second strand VI, in an annealing step. As 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. Referring to Fig. IE, following the elongation step, two new DNA molecules VII and VIII are synthesised, molecule VII comprising the tail sequence 10 in a single stranded forms, and molecule VIII comprising a double stranded tail region, with the tail sequence 10 having a complementary tail region 12.

Referring to Fig. IF, 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. As illustrated in Fig. 1G, the probe 5 will therefore bind to the tail region 12 of molecule IX. During the elongation step, 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.

Referring to Fig. II, 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. In the next embodiment, 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.

Referring to 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.

Referring initially to Fig. 2A, there is illustrated 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 30B 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 50A having a first fluorescent label 60A, a quencher 70, and a first probe sequence 80A, and a second probe 50B having a second fluorescent label 60B, a quencher 70, and a second probe sequence 80B. The first forward PCR primer 20A additionally comprises a tail sequence 100A 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.

Referring to Figs. 2B and 2C, 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 100A (Fig. 2C).

Referring to Fig. 2D, 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 20A binds to the 5’ end of strand VI, and the common reverse PCR primer 40 binds to the 5’ end of the second strand VI, in an annealing step. As 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. 2E, following the elongation step, two new DNA molecules VIII and IX are synthesised, molecule VIII comprising the tail sequence 100A in a single stranded forms, and molecule IX comprising a double stranded tail region, with the tail sequence 100A having a complementary tail region 120A.

Referring to Fig. 2F, a third round of PCR is initiated with a denaturation step and the DNA molecule IX denaturing to form single stranded DNA molecules X and XI. Single stranded DNA molecule XI comprises a tail region 120A that is complementary to the tail region 100A of probe 50A, and therefore complementary to the probe sequence 80A. As illustrated in Fig. 2G, the probe 50A will therefore bind to the tail region 120A of molecule XI. 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.

Experimental examples Example 1 Experimental description: In the following example, samples that were previously tested and assigned SNP genotypes using KASP chemistry were selected to be re tested with the IDENTISNP™ 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 IDENTISNP™ chemistry. In order to assess the success of the IDENTISNP™ 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 95°C 5 minutes 1 cycle 95°C 10 seconds 40 cycles 60°C 60 seconds Table 1: Specific PCR cycling conditions for IDENTISNP™ chemistry Results 40 Cycles 45 Cycles 50 cycles Change in call rates N/A -0.99% -1.18% Table 2. Call rate stability with IDENTISNP™ during re-cycling process IDENTISNP™ 1-cycle IDENTISNP™ 3-cycle SNP genotypes compared 16,494 16,770 % concordance 99.67% 99.64% Table 3. Concordance rates of KASP vs IDENTISNP™ - KASP scored independently and in its recommended fashion of multiple read analysis No. of samples used % SNPs typed Assignment % Assignment concordance with KASP Female Parents 80 99.24 N/A N/A Male Parents 80 97.77 N/A N/A Offspring 220 98.51 100 100 Table 4. Mendelian inheritance - IDENTISNP™ SNP call % and parentage assignment concordance with KASP Conclusions Samples previously tested with the KASP chemistry and analysed to assign parentage where re-tested using IDENTISNP™ single read and multiple read to see would the parentage assignment be equivalent to the previous KASP assignments.

In Table 2 it should be noted that unlike KASP none of the sample calls changed to a different call with over cycling, i.e. 100% concordance. Differences in call rates are down to rigidity of analyst calling. Unlike KASP where over cycling leads to call changes from 3 distinct groups into unclear scattergun plots or all samples merging into a single heterozygous called group, making a single read call difficult and inaccurate if sample quality and concentration is not consistent. It is a costly, if not sometimes impossible exercise, to insure high sample quality, as well as to normalise DNA concentration. Thus far these issues have not given cause for concern with the IDENTISNP™ chemistry exercise.

SNP concordance rate with KASP is >99% in both the single read and multiple read analysis using the IDENTISNP™ chemistry (see Table 3.).

It can be concluded from these results that in real world, practical applications, the IDENTISNP™ chemistry single read is at least the equivalent of the KASP chemistry re cycling protocol.

Example 2 Experimental description 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.

Experimental procedure (brief) 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 IDENTISNP™ 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.

Once the PCR protocol had been run, and the alleles assigned, analysis of the SNP genotyping data obtained with the IDENTISNP™ chemistry was completed by our bioinformatics department. The results of the SNP genotyping were analysed using our in house data analysis software (there are several similar freeware types of this software online) that assigns parents to each offspring based on Mendelian inheritance of the specific SNPs that were genotyped.

We compare the results of this data to a similar project that had been previously run for the same purpose using solely KASP chemistry. While not a direct comparison it is an equivalent one.

Results Summary For the IDENTISNP™ chemistry, analysis of the results shows us that male parents were generally mated to ~3 females, and females to ~2 males Parentage test result No. offspring % of offspring Both parents assigned 3006 91.6% Female parent assigned 94 2.9% Male parent assigned 63 1.9% No parents assigned 77 2.3% QC fail 43 1.3% Total 3283 Table 5. Results of parentage assignment with IDENTISNP™ chemistry In comparison to a similar genotyping experiment carried out with KASP where male parents were generally mated to ~2 females, and some females to multiple males Parentage test result No. offspring % of offspring Both parents assigned 4192 90.7% Female parent assigned 312 6.8% Male parent assigned 2 0.0% No parents assigned 23 0.5% QC fail 92 2.0% Total 4621 Table 6. Results of parentage assignment with KASP chemistry Error rates Description of error rate % error (SNP calls) per locus Mendelian error rate 0.016153846 Highest per locus % error 0.7 per offspring Mendelian error rate 0.017957635 Highest per offspring % error 3.1 Table 7. IDENTISNP™ chemistry error rates Description of error rate % error (SNP calls) per locus Mendelian error rate 0.130137 Highest per locus % error 3.36 per offspring Mendelian error rate 0.160393812 Highest per offspring % error 3.3 Table 8. KASP chemistry error rates Conclusion Samples that were never previously tested were tested with the IDENTISNP™ chemistry and then analysed to assign parents to offspring as trios. Calls were made using a single read and analysis also acknowledged any Mendelian errors that may have occurred. Errors such as this are generally due to miscalls of a SNP in either an offspring, leading to minor assignment issues, or in a parent, which can lead to large scale assignment issues as the error will affect all assignments of offspring to that parent.

In the IDENTISNP™ project (Table 5.) there were 33 offspring samples, from the 3163 assigned samples (Both parents assigned, female parents assigned and male parents assigned) with Mendelian errors (1.04%). This compares to the equivalent KASP project (table 6.) which had 395 offspring samples, from the 4503 assigned samples (Both parents assigned, female parents assigned and male parents assigned) with Mendelian errors (8.77%). Using our accepted threshold of allowing 2 errors per sample, we find that IDENTISNP™ had 3 unacceptable samples, 0.095%, compared to KASP which had 8 unacceptable samples, 0.178%.

Results in Table 5 and 6 are divided into five categories. From this data we see that both IDENTISNP™ and KASP had comparable assignments of both parents to their offspring with IDENTISNP™ slightly higher at 91.6% compared to KASPs 90.7%.

Again this data confirms that the IDENTISNP™ chemistry is at least comparable to the current KASP chemistry.

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 IDENTISNP™ single read. On average there has been a reduction in scoring time of circa 75%, with times varying between analysts and data sets.

It can be concluded from these results that in real world, practical applications, the IDENTISNP™ 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 Experimental description The aim of this examination is to compare customer data in routine use which has used both chemistries. While we have run several comparative projects to compare the two chemistry types directly, we accept that these may appear to have the possibility of bias in favour of IDENTISNP™. To this end we compared large dataset over time between IDENTISNP™ and KASP, where KASP was scored before the invention of IDENTISNP™, and both are scored with commercial concerns.

Experimental procedure (brief) 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 1st January 2014 until the 31st of March 2014 provided by KASP, and data from the 1st January 2015 until the 31st of March 2015 provided by IDENTISNP™. 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 IDENTISNP™ was run with the protocol detailed in Table 12.

Stage Temperature Time No. of cycles Hot Start 94°C 15 minutes 1 Touchdown 94°C 20 seconds 10 65°C (-0.8°C per cycle) 60 seconds Amplification 94°C 20 seconds 26 57°C 60 seconds Read Stage 30°C 60 seconds 1 Amplification 94°C 20 seconds 3 57°C 60 seconds Read Stage 30°C 60 seconds 1 Amplification 94°C 20 seconds 26 57°C 60 seconds Read Stage 30°C 60 seconds 1 Table 9. KASP protocol (taken from “KASP thermal cycling conditions” by LGC Genomics).

Stage Temperature Time No. of cycles Hot Start 94°C 120 seconds 1 Amplification 94°C 10 seconds 40 60°C 60 seconds Read Stage n/a 60 seconds 1 Table 10. IDENTISNP™ protocol (taken from “IdentiSNP user manual vl.O”) We compared the results of the data from both time periods, including genotyping call rate, 5 Quality Control pass rate (>70% genotyping call rate) to assess if there has been any improvements or deterioration in terms of data quality between the two.

Results Summary Porcine samples comparison Chemistry Genotype data points QC pass rate (>70%) QC pass rate difference Genotyping % Genotyping % difference KASP 101760 81.04% - 81.04% - IDENTISNPT M 144384 83.67% + 2.63% 83.67% + 2.63% Table 11. Porcine sample genotype rate comparison over 3 months Bovine samples comparison Chemistry Genotype data points QC pass rate (>70%) QC pass rate difference Genotyping % Genotyping % difference KASP 1386520 88.84% 85.27% - IDENT1SNPT M 1845840 95.33% + 6.49% 90.51% + 5.24% Table 12. Bovine sample genotype rate comparison over 3 months Salmon samples comparison Chemistry Genotype data points QC pass rate (>70%) QC pass rate difference Genotyping % Genotyping % difference KASP 1338047 89.59% 88.96% IDENTISNPT M 982245 93.80% + 4.21% 91.86% + 2.91% Table 13. Bovine sample genotype rate comparison over 3 months Template Chemistry No. of genotype data points Call rate % difference Salmon KASP 1338047 - Salmon IDENTISNP™ 982245 + 2.91% Porcine KASP 101760 - Porcine IDENTISNP™ 144384 + 2.63% Bovine KASP 1386520 - Bovine IDENTISNP™ 1845840 + 5.24% Table 14. Id lentiSNP vs KASP cal] rate comparison (QI 2014 vs Q2 2015) Conclusion 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.

In all 3 comparisons, both the genotyping call rate and the sample quality control rate was higher for IDENTISNP™ than KASP as can be seen in Tables 11,12 and 13. Due to the large number of data points, it is clear, that there is a statistically significant difference between the two chemistries in terms of genotyping call rate. This is detailed fully within Table 14.

Tables 9 and 10 detail the comparable PCR protocols for IDENTISNP™ and KASP, when the direct PCR time is calculated (3840 seconds for KASP, 2980 seconds for IDENTISNP™), 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 IDENTISNP™ 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. This adds on a minimum of 600 seconds (plus additional machinery handling time).

Following on from this, data analysis upon review has been seen to be reduced by up to 75%, where in the removal or “recycling” and data point “tracking” inherent in KASP leads to a significant reduction in data analysis and review.

The invention is not limited to the embodiments herein before described which may be varied 5 in construction and detail without departing from the spirit of the invention.

Claims (5)

1. 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; - a reverse PCR primer having a sequence that is complementary to the target double stranded nucleic acid; and - 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 method comprising the steps of: - incubating the forward and reverse PCR primers, dual labelled probe, and sample together; - performing at least two rounds of PCR to generate a double stranded nucleic acid comprising the tail region and a sequence complementary to the tail region; and - initiating a further round of PCR in which the 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.

2. 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 reverse PCR primer having a sequence that is complementary to the target double stranded nucleic acid; and - 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,

3. 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 forward PCR primer having a first allele-specific sequence and a first tail sequence; - a second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence; - a common reverse PCR primer; - 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; and - 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 method comprising the steps of: - incubating the forward and reverse PCR primers, probes, and sample together; - initiating at least two rounds of PCR to generate a double stranded nucleic acid comprising the first or second tail region and a sequence complementary to the first or second tail region; - initialling a further round of PCR in which one of the probes binds to the sequence complementary to the first or second tail region, whereby the oligonucleotide probe is displaced and cleaved resulting in a detectable signal from the reporter label; and - correlating the detectable signal from the reporter label with the presence of the first or second allele of the locus in the sample of DNA.

4. 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 forward PCR primer having a first allele-specific sequence and a first tail sequence; - a second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence; - a common reverse PCR primer; - 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; and - 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 method comprising the steps of: - incubating the forward and reverse PCR primers, probes, and sample DNA together; - initiating at least two rounds of PCR to generate a double stranded nucleic acid comprising the first or second tail region and a sequence complementary to the first or second tail region; - initialling a further round of PCR in which one of the probes binds to the sequence complementary to the first or second tail region, whereby the oligonucleotide probe is displaced and cleaved resulting in a detectable signal from the reporter label; and - correlating the detectable signal from the reporter label with the presence of the first or second allele of the sample of DNA (i.e. presence or absence of the SNP).

5. A kit suitable for genotyping a sample of DNA for the presence or absence of a single nucleotide polymorphism within the DNA, the kit comprising: - a first forward PCR primer having a first allele-specific sequence and a first tail sequence; - a second forward PCR primer having a second allele specific sequence and a second tail sequence different to the first tail sequence; - a common reverse PCR primer; - 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; and - 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.