JP6853523B2 - PCR using helicase - Google Patents

Description

The present invention relates to PCR using helicase.

Digital PCR has been attracting attention in recent years as a method capable of performing absolute quantification and detection of rare alleles without depending on the number of amplification cycles when measuring the initial amount of nucleic acid in each sample. For example, it is considered to be useful for genetic testing, especially applications such as CNV (copy number variation) analysis, Real sequence detection, and gene expression analysis. Research and development related to digital PCR is also being vigorously promoted (for example, Patent Documents 1 to 3).

In digital PCR, for example, a nucleic acid sample (for example, a sample containing a target nucleic acid such as DNA or cDNA) is diluted as necessary, distributed to a large number of wells, and PCR is performed in each well (in parallel). .. At this time, some wells contain one or more target molecules, and some wells do not contain any target molecules. After the completion of PCR, the presence or absence of PCR amplification is analyzed for each well, and the number of wells with amplification is counted as a positive reaction containing the target molecule, and the number of wells without amplification is counted as a negative reaction not containing the target. By detecting the presence or absence of a signal in each well after PCR in this way, it is possible to absolutely quantify the target molecule in the sample based on the ratio of positive reaction and / or negative reaction. No comparison with standard or control samples is required. Since the target molecules in the sample are randomly distributed, there is no certainty that only one molecule has been distributed to the wells that have been positively reacted. Therefore, in order to calculate the wells in which a plurality of target nucleic acids may be distributed, it is possible to apply a correction coefficient by statistical processing (for example, processing using a Poisson model) to calculate the absolute number of target nucleic acids. it can.

As described above, since digital PCR is a technique for performing absolute quantification of the target nucleic acid depending on whether nucleic acid amplification occurs in the well or not, false positive (that is, amplification in the well containing no target nucleic acid (for example, non-specific amplification)) is performed. The presence of (what is seen) can greatly impair its accuracy. In particular, when the volume of the reaction solution is large (for example, 1 μL or more), the band of the amplification product is analyzed by electrophoresis of the reaction solution, and the band is a specific amplification product or a non-specific amplification product. However, when the volume of the reaction solution is small (for example, less than 1 μL) as in digital PCR, such analysis cannot be performed, so that particularly high accuracy and reaction efficiency are required. Therefore, in PCR with a small reaction solution volume such as digital PCR, it is required to suppress false positives in order to improve accuracy as much as possible.

International Publication No. 2009/033178 International Publication No. 2013/1138889 International Publication No. 2014/043581 International Publication No. 2016/013620

An object of the present invention is to improve the accuracy of digital PCR, particularly to suppress false positives in digital PCR.

The present inventors have found that by including helicase in the PCR reaction solution, false positives can be suppressed in PCR using a very small amount (for example, nanoliter order or less) of the PCR reaction solution, and further improvements have been made to the present invention. The invention was completed.

The present invention includes, for example, the subjects described in the following sections.
Item 1.
Helicase-containing PCR mix dispensed into 0.05-500 nL.
Item 2.
A PCR mix containing helicase for digital PCR.
Item 3.
A helicase-containing PCR mix dispensed into 0.1-500 nL for digital PCR.
Item 4.
The PCR mix according to any one of claims 1 to 3, wherein the helicase is at least one selected from the group consisting of TK0566 and TK0178 and variants thereof.
Item 5.
The PCR mix according to any one of claims 1 to 4, wherein the helicase concentration is 100 nM or less.
Item 6.
A PCR kit comprising the PCR mix according to any one of claims 1 to 5 or for preparing the PCR mix according to any one of claims 1 to 5.
Item 7.
A method for amplifying nucleic acid by PCR (preferably digital PCR) using the PCR mix according to any one of claims 1 to 5. (Preferably, the method of amplifying nucleic acid by PCR using the PCR mix according to any one of claims 1 to 5 and having an annealing temperature of 55 to 63 ° C.).

According to the present invention, false positives can be suppressed even in PCR using a very small amount (for example, nanoliter order or less) of PCR reaction solution.

The sequence of the probe (PCR primer) used in the example is shown. The fluorescent dye VIC is bound to the probe whose underlined portion is G, and the fluorescent dye FAM is bound to the probe whose A is A. In the example, the distribution of fluorescence of each well when digital PCR is performed at an annealing temperature of 56 ° C. is shown. In the example, the distribution of fluorescence of each well when digital PCR is performed at an annealing temperature of 60 ° C. is shown.

Hereinafter, each embodiment of the present invention will be described in more detail.

The present invention includes a helicase-containing PCR mix that has been dispensed in trace amounts. The PCR mix is preferably dispensed into 0.05 to 500 nL, more preferably 0.1 to 100 nL, and more preferably 0.5 to 50 nL. preferable.

The PCR mix refers to a solution (particularly an aqueous solution) containing at least one component necessary for the PCR reaction in which the target nucleic acid is amplified. The PCR mix is a PCR reaction solution (which contains all the components necessary for the PCR reaction amplified by the target nucleic acid, and PCR can be performed if the temperature is changed appropriately) or a solution in the middle of preparing the PCR reaction solution (temperature). Although PCR does not occur even if the amount is changed, one or more components necessary for the PCR reaction are contained). The components required for PCR are components in which PCR does not occur in which the target nucleic acid is amplified if the component is absent, and polymerases (particularly DNA / RNA polymerases), dNTPs (dATP, dTTP, dGTP, dCTP), oligonucleotides (primers). ), Target nucleic acid (DNA or RNA). The content of these components can be appropriately set. Although not particularly limited, for example, the primer concentration in the mix is preferably about 0.05 to 0.5 μM, more preferably about 0.1 to 0.4 μM, and more preferably about 0.1 to 0.3 μM. Is even more preferable.

The PCR mix of the present invention contains helicase (that is, helicase-containing PCR mix). The target nucleic acid may be any nucleic acid that acts as a so-called template, and a nucleic acid having an amplification target nucleic acid is preferable. Further, among the nucleic acids, DNA is preferable.

As the helicase contained in the PCR mix, a known helicase can be used, and for example, a DEAD-box type RNA helicase derived from hyperthermic archaea and a variant thereof can be used. More specifically, for example, the DEAD-box type RNA helicase derived from hyperthermic archaea described in International Publication No. 2016/013620 and its variants can be used.

Moreover, TK0566 and TK0178 and their variants can be used particularly preferably. Both TK0566 and TK0178 are helicases derived from the thermophile Thermococcus kodakalensis belonging to the genus Thermococcus. The amino acid sequence of TK0566 is shown in Sequence 1 and the amino acid sequence of TK0178 is shown in Sequence 2. The variant here means one having a high identity and helicase activity, although the amino acid sequences are not the same. A variant of TK0566 is a helicase consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of TK0566, and which has helicase activity. A variant of TK0178 is a helicase that consists of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of TK0178, and has helicase activity. Here, "1 or 2 or more" in "1 or 2 or more amino acids are deleted, substituted or added" is preferably 1 to 30 (that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30), more preferably. It refers to 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1, 2 or 3. The amino acid sequence of the variant of TK0566 preferably has 80% or more identity with the amino acid sequence of TK0566, more preferably 90% or more, further preferably 95% or more, and 98% or more. Is even more preferable, and 99% or more is particularly preferable. The amino acid sequence of the variant of TK0178 preferably has an identity of 80% or more, more preferably 90% or more, further preferably 95% or more, and 98% or more with the amino acid sequence of TK0178. Is even more preferable, and 99% or more is particularly preferable.

In addition, in this specification, having a helicase activity means an activity of destabilizing a spiral of a double-stranded nucleic acid by using the energy of ATP hydrolysis and rewinding the double-stranded nucleic acid into a single-stranded nucleic acid. Say. The unwinding activity and ATP hydrolysis activity of the double-stranded nucleic acid can be measured by the method described in International Publication No. 2016/013620.

The helicase content ratio (concentration) in the helicase-containing PCR mix can be appropriately set and is usually 100 nM or less, for example, 1 to 100 nM is preferable, 5 to 75 nM is more preferable, and 10 to 50 nM is further preferable. Since the efficiency of suppressing false positives in PCR may vary depending on the helicase type and its content ratio (concentration), it may be important to adjust the helicase type and its content ratio (concentration). In particular, when TK0566 or a variant thereof is used as the helicase, 10 to 30 nM is preferable. In particular, when TK0178 or a variant thereof is used as the helicase, 15 to 45 nM is preferable.

The helicase-containing PCR mix (particularly the PCR reaction solution) can be particularly preferably used for digital PCR. In addition, when it is necessary to carry out PCR using a very small amount of PCR mix, it can be preferably used for that PCR.

The present invention also includes a method of amplifying nucleic acid by performing PCR using the above-mentioned helicase-containing PCR mix (particularly PCR reaction solution). Reaction conditions such as the PCR temperature cycle (denaturation, annealing, and extension reaction temperatures) and the number of cycles can be appropriately set. For example, denaturation can be performed at 90 to 97 ° C. for 10 to 50 seconds, annealing can be performed at 50 to 65 ° C. for 1 to 5 minutes, and the extension reaction temperature can be performed at 65 to 75 ° C. for 1 to 5 minutes. Further, as the number of cycles, for example, about 35 to 45 cycles can be mentioned.

In order to suppress false positives in PCR, it is important to adjust the annealing temperature among various conditions. The annealing temperature depends on the primer used, but is particularly preferably about 55 to 63 ° C. (55, 56, 57, 58, 59, 60, 61, 62, or 63 ° C.).

The present invention also includes (i) a PCR kit comprising the helicase-containing PCR mix, or (ii) a PCR kit for preparing the helicase-containing PCR mix. The kit (i) includes a helicase-containing PCR mix (either a PCR reaction solution or a PCR reaction solution preparation intermediate solution), and is also provided with instruments and / or reagents necessary or convenient for PCR. The instruments and reagents provided in the kit (i) include PCR tubes, PCR microchips, helicases (particularly TK0566 or TK0178, or variants thereof), polymerases (particularly DNA / RNA polymerases), dNTPs (dATP, dTTP). , DGTP, dCTP), oligonucleotides (forward primer and / or reverse primer), amplification target nucleic acid (DNA or RNA) and the like, but are not particularly limited. The primer used in the present invention is preferably labeled, and examples of the label include a fluorescent label and an RI (radioisotope) label. Further, the kit (ii) is a kit provided with an instrument and / or a reagent for preparing the helicase-containing PCR mix (particularly a PCR reaction solution), and the helicase using the instrument and / or the reagent. It is a kit that can prepare the contained PCR mix. As the instruments and reagents obtained in the kit of (ii), for example, the same instruments and reagents as those provided in the kit of (i) above can be used.

In addition, in this specification, "including" also includes "consisting essentially" and "consisting of" (The term "comprising" includes "consisting essentially of" and "consisting of.").

Hereinafter, the present invention will be specifically described, but the present invention is not limited to the following examples. TK0566 was also expressed and purified by the method described in Examples of WO 2016/013620. In addition, TK0178 is a method for expressing and purifying TK0566, except that the primer set used for amplifying the TK0178 gene (see Table 1 below) and the restriction enzymes for treating the amplified nucleic acid were changed to BamHI and NotI. Prepared in the same manner as above.

Examination of the effect of helicase on digital PCR The following helicase, target nucleic acid (template nucleic acid), and probe (PCR primer set for SNP detection) are added to Quant Studio ^{TM 3D Digital PCR Master Mix V2 (Thermo Fisher), and the PCR mix used for the examination.} Was prepared. The polymerase is included in the Master Mix.

Helicase: TK0566 or TK0178 with a final concentration of 0, 20, or 40 nM
Target nucleic acid (human genomic DNA): 5 ng
Probe (PCR primer): C____1202883_20 returned 1 TaqMan SNP Genotyping Assay (Thermo Fisher) with a final concentration of 0.2 μM

Then, using the obtained PCR mix, digital PCR was performed under the following reaction conditions.
Temperature conditions: "95 ° C for 30 seconds, 56 ° C or 60 ° C for 2 minutes, 72 ° C for 30 seconds" for 39 cycles Digital PCR device: Digital PCR 3D (Thermo Fisher Scientific)
Chip for digital PCR: 20,000-well chip

The amount of PCR mix prepared was 14.5 μL, which was equally dispensed into 20,000 wells. Therefore, 0.725 nL was dispensed per well. The probe used (C____1202883_20) is a probe for detecting the SNP (ID: rs1801133) of the methylenetrahydrofolate redutase (NAD (P) H) gene. The sequence of the probe (TaqMan Probe) labeled with VIC or FAM is shown in FIG. The probe that detects C of SNP (GAAAAGCTGCGTGATAGAAATCG [G] CTCCCGCAGACACTTCCTTCTACA) has a fluorescent dye VIC, and the probe that is T of SNP (GAAAAGCTGCGTGATGATGAAATCCG [A] CTCCCGCAGACTCA) has a fluorescent dye VIC. Therefore, the well in which the fluorescence of VIC is detected by digital PCR contains DNA having SNP C, and the well in which fluorescence of FAM is detected contains DNA having SNP T. I understand. In addition, C____1202883_20 also includes a reverse primer capable of nucleic acid amplification using the two types of fluorescent dye-labeled probes as forward primers.

The results at the annealing temperature of 56 ° C. are shown in FIG. In both TK0566 and TK0178, the addition of 20 nM to the reaction solution increased the fluorescence intensity of the positive sample, and an improvement in the positive and negative determination was observed.

The results at an annealing temperature of 60 ° C. are shown in FIG. Similar improvement was observed by adding 20 nM of TK0566 to the reaction solution. However, no such improvement was observed when 40 nM was added. Further, when 20 nM of TK0178 was added to the reaction solution, the same improvement was observed, and when 40 nM was added, the same improvement was remarkably observed.

2 and 3 are views in which the fluorescence intensity in each well was measured and plotted according to the VIC fluorescence intensity (horizontal axis) and the FAM fluorescence intensity (vertical axis). Each plotted point is color-coded in four colors (yellow, blue, red, and green). Red is the well where VIC fluorescence was detected and FAM fluorescence was not detected, blue is the well where FAM fluorescence was detected and VIC fluorescence was not detected, and yellow is the well where neither VIC nor FAM fluorescence was detected. Wells and greens indicate wells in which both VIC and FAM fluorescence were detected. The distribution of these color-coded points is such that the points of the same color are concentrated in one place as much as possible, and the more the points of different colors are separated as much as possible, the easier it is to distinguish the presence or absence of amplification of the target nucleic acid. Therefore, false positives can be reduced.

Claims (6)

A PCR mix containing helicase, dispensed into 0.05-500 nL .
The helicase concentration is 100 nM or less,
The helicase has TK0566, which is a helicase consisting of the amino acid sequence shown in SEQ ID NO: 1, TK0178, which is a helicase consisting of the amino acid sequence shown in SEQ ID NO: 2, and TK0178 which is 90% or more identical to these amino acid sequences and has helicase activity. At least one selected from the group consisting of variants,
PCR mix . The PCR mix according to claim 1 , which is for digital PCR. The PCR mix according to claim 1 or 2, wherein the helicase concentration is 5 to 75 nM or less . The PCR mix according to any one of claims 1 to 3, which has been dispensed into 0.1 to 100 nL. Comprising a PCR mix according to any one of claims 1-4, or, for the preparation of PCR mix according to any one of claims 1-4, PCR kits. A method for amplifying nucleic acid by PCR using the PCR mix according to any one of claims 1 to 4.

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