JP6565147B2 - Multiplex PCR method - Google Patents

Description

The present invention relates to the field of nucleic acid amplification. More specifically, the present invention relates to a multiplex PCR method.

Synthesis of DNA from a template nucleic acid using a DNA polymerase is used and applied to various methods such as sequencing and nucleic acid amplification in the field of molecular biology. Among them, the nucleic acid amplification method has already been put into practical use not only in the research field, but also in the forensic field such as genetic diagnosis and parentage testing, or in the inspection of microorganisms in food and the environment.

  Numerous nucleic acid amplification methods have been developed to date, such as polymerase chain reaction (PCR) method, Loop-Mediated Isotropic Amplification (LAMP) method, Transcribation Reverse Transfer-Concluded Reaction Reaction (TRC) SN method. Nucleic acid amplification methods such as Amplification (NASBA) are relatively popular.

Among these, the PCR method used for amplification of a specific sequence of DNA is a technique that is very widespread from research fields to application fields. Currently, the PCR method is being further developed, and there are various technologies such as the Multiplex PCR method that simultaneously amplifies multiple primers, and the real-time PCR method that detects fluorescent amplification products in real time using a fluorescent dye. To do. These techniques have already been put into practical use not only in the research field, but also in the forensic field, food, and environmental microbiological tests.

The gene amplification method by the PCR method is performed by repeating 20 to 50 cycles consisting of denaturation, annealing, and extension in the presence of a target nucleic acid, four types of deoxynucleoside triphosphates, at least a pair of primers and a DNA polymerase. This is a method of exponentially amplifying a target nucleic acid region sandwiched between a pair of primers (Patent Document 1).

In general, the PCR method is performed by adding a pair of primer sets. However, when it is desired to detect amplification products at the same time, when it is desired to save reagents, equipment and time, and when the amount of template sample is limited, a PCR method that simultaneously amplifies a plurality of target regions using a large number of primer sets, so-called A multiplex PCR method is performed (Non-patent Document 1).

In the multiplex PCR method, a multiplex PCR of a short chain of 1 kb or less is generally performed. However, recently, multiplex PCR has been performed for long-chain targets of 1 kb or more in the next-generation sequencer (NGS) resequencing analysis, and the demand for long-chain target multiplex PCR is increasing. .

  However, in multiplex PCR, there are problems that non-specific amplification and primer dimers are likely to occur, and that the amplification efficiency of each target tends to be different. Further, the longer the target, the higher the possibility that the target sequence contains a difficult sequence (high GC content or high AT content), and the amplification success rate when an arbitrary target region is amplified decreases. There has been a demand for multiplex PCR that solves these problems.

JP-A-2005-328709 Japanese Patent Application No. 2012-116926

M. C. Edwards and R. A. Gibbs, Genome Res. , 3, 65-75 (1994) Alan H. Beggs et al. , Human Genetics, 86, 45-48 (1990).

The present invention has been made against the background of such prior art problems. That is, an object of the present invention is to provide a multiplex PCR method that suppresses the generation of non-specific amplification and primer dimers and is unlikely to give a difference in the amplification efficiency of each target.

As a result of intensive investigations, the present inventors have found that the use of a DNA polymerase belonging to Family B not only causes less non-specific amplification and primer dimer, but also has a high amplification success rate for long-chain targets, and is included in the target. The inventors have found that multiplex PCR that is difficult to be subjected to amplification inhibition can be performed even if components coexist, and have found that the above problems can be solved based on this finding, and have reached the present invention.

That is, the present invention has the following configuration.
Item 1.
A method for specifically amplifying two or more target regions with one reaction solution, wherein the reaction solution contains a DNA polymerase belonging to Family B.
Item 2.
Item 2. The method according to Item 1, wherein the target region includes at least one target region of 1 kb or more.
Item 3.
Item 2. The method according to Item 1, wherein the target region includes at least five target regions of 10 kb or more.
Item 4.
Item 4. The method according to Items 1 to 3, wherein the target region is a region of 10 or more locations.
Item 5.
Item 5. The method according to Item 1 to 4, wherein the DNA polymerase belonging to Family B is derived from Thermococcus kodakaraensis.
Item 6.
Item 6. The method according to Items 1 to 5, wherein a biological sample that has not undergone a purification step is added to a nucleic acid amplification reaction to amplify a target nucleic acid in the biological sample.
Item 7.
Item 7. The method according to Item 6, wherein the biological sample is blood.
Item 8.
Item 8. The method according to Item 1 to 7, wherein the copy number bias of the amplification product is within 100 times.

According to the present invention, the range of amplification lengths applied to multiplex PCR can be expanded and amplified uniformly. Furthermore, multiplex PCR is possible from biological samples such as blood without pretreatment.

It is the figure which evaluated multiplex PCR of the range of 100 bp-1 kb. It is the figure which evaluated multiplex PCR of the range of 1 kb-10 kb. It is the figure which evaluated multiplex PCR of the range of 100 bp-1 kb using blood as a template. It is the figure which evaluated multiplex PCR of the range of 1 kb-10 kb using blood as a template. It is a figure showing the calibration curve created with respect to the target of 10 kb. It is a figure showing the calibration curve created with respect to the target of 10 kb. It is a figure showing the calibration curve created with respect to the target of 10 kb. It is a figure showing the calibration curve created with respect to the target of 10 kb. It is the figure which evaluated the copy number and bias of the multiplex PCR amplification product of 10kbx10 target.

Hereinafter, the present invention will be described in detail.
One embodiment of the present invention is a method for specifically amplifying two or more target regions with one reaction solution, wherein the reaction solution contains a DNA polymerase belonging to family B. It is.

A “target region” is a target nucleic acid sequence region that is amplified by a primer set. In the method of the present invention, the length of the target target is not limited, but multiplex PCR can be performed on a long-chain target including a region of 1 kb or more, and further 10 kb or more. In the method of the present invention, the number of target targets is not particularly limited as long as it is 2 or more, but multiplex PCR can be performed even if it is 5 or more, and even 10 or more. The upper limit of the number of targets is not particularly limited, but is preferably 1000 or less.

The method of the present invention is characterized in that a DNA polymerase belonging to Family B is used as a DNA polymerase in the multiplex PCR method.
In general, a DNA polymerase belonging to Family A has been used for PCR because of its high amplification efficiency and easy setting of conditions. However, recently, conditions can be easily set even for a DNA polymerase belonging to Family B by various studies such as reaction composition. DNA polymerases belonging to family B are characterized by high accuracy, high thermal stability, and resistance to inhibitors introduced into the reaction system.

  The DNA polymerase belonging to Family B in the present invention is not particularly limited, but is preferably a DNA polymerase derived from Archaea. Archaea-derived DNA polymerases belong to either Family B Pol I type or Pol II type. In the present invention, a DNA polymerase belonging to Pol I type is preferable. Examples of DNA polymerases derived from archaea belonging to the family B Pol I type include DNA polymerases isolated from bacteria of the genus Pyrococcus and Thermococcus. Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus friosus, Pyrococcus sp. Including, but not limited to, DNA polymerase isolated from GB-D, Pyrococcus Woesii, Prococcus abysii, Prococcus horikoshii. Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermocoss gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcsus sp. 9 degree North-7 (Thermococcus sp. 9 ° N-7), Thermococcsus sp. Including but not limited to DNA polymerase isolated from KS-1, Thermococcus celler, or Thermococcus sicili. PCR enzymes using these DNA polymerases are commercially available, such as Pfu (Staragene), KOD (from Toyobo, Thermococcus kodakaraensis), Pfx (Life Technologies BioNevels), Vent (New England BioLabs). England Biolabs), Tgo (Roche), Pwo (Roche) and the like. KOD polymerase is preferred.

  Other configurations of the PCR reaction solution in the method of the present invention are not particularly limited, but include a heat-resistant DNA polymerase, a template nucleic acid, one or more oligonucleotide primers, one or more deoxynucleotide triphosphates, or deoxynucleotide three. It is preferable to contain at least one selected from the group consisting of phosphoric acid derivatives, buffers, and salts.

  In the method of the present invention, the nucleic acid used as a template is not particularly limited. Examples include synthetic DNA, genomic DNA purified from biological samples, and cDNA obtained from RNA by reverse transcription. Further, there is no need for purification, and a crude sample of a biological sample containing a nucleic acid as a template or the biological sample itself can be used.

The one or more oligonucleotide primers used in the present invention are oligonucleotides complementary to each nucleic acid strand of the nucleic acid to be amplified, and it is preferable to use two or more primers. The primer is substantially complementary to each strand of a double-stranded nucleic acid, and when the extension product synthesized from one primer is separated from its complement, the extension product is It is chosen so that it can serve as a template for the synthesis of the extension product of the other primer.

  One or more deoxynucleotide triphosphates or derivatives of deoxynucleotide triphosphates are used as substrates in PCR reactions. As this substrate, four kinds of deoxyribonucleoside triphosphate (dNTPs; a mixture of dATP, dCTP, dTTP and dGTP) solution and the like may be contained.

It is preferable that the PCR reaction solution further contains a buffer. Examples of the buffer include Tris (TRIS), Tricine (TRICINE), Bis-Tricine (BIS-TRICINE), Hepes (HEPES), Mops (MOPS), Tes (TES), Tapes (TAPS), Pipes (PIPES). ), And caps (CAPS), but are not particularly limited. As a density | concentration, about 10-200 mM is preferable and about 20-100 mM is more preferable. As pH, the range of about 7.0-9.5 is preferable, and the range of about 7.5-9.0 is more preferable.
The buffer solution preferably contains Mg ²⁺ at a concentration of about 1 to 5 mM, preferably about 1.5 to 2.5 mM. Furthermore, KCl may be included.

  Further, the PCR reaction solution may contain albumin, glycerol, heparin, trehalose, betaine and the like. What is necessary is just to add these addition ratios in the range which does not inhibit PCR reaction.

  In the present invention, the nucleic acid amplification reaction may be performed in the presence of a single-stranded binding protein or a mismatch binding protein.

The bias represents a difference in amplification amount between a target with the largest amplification amount and a target with the smallest amplification amount among amplification products in multiplex PCR. The greater the bias, the greater the difference in amplification. The difference in the amplification amount in the method of the present invention is not particularly limited, but the amplification amount of the target with the largest amplification amount is preferably within 300 times the amplification amount of the target with the smallest amplification amount, and within 100 times. Is more preferable, and is more preferably 50 times or less.

<Evaluation method of multiplex PCR (target length: 100 bp to 1 kb)>
PCR uses Buffer, dNTPs, enzyme solution (KOD FX Neo) attached to KOD FX Neo (manufactured by Toyobo, using KOD polymerase), 1 × PCR Buffer, and 15 pmol of each primer (SEQ ID NOs: 1 to 18, Non-patent document 2), 50 μl of a reaction solution containing 50 ng of human genomic DNA (Roche) and 1 U of KOD FX Neo is prepared. The reaction is carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 60 ° C., 30 seconds → 68 ° C., 1 minute.
After completion of the reaction, 5 μl of the reaction solution is subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the approximately amplified DNA fragment is confirmed under ultraviolet irradiation.

<Evaluation method of multiplex PCR (target length: 1 kb to 10 kb)>
PCR was performed using Buffer, dNTPs, enzyme solution (KOD FX Neo) attached to KOD FX Neo (Toyobo), 1 × PCR Buffer, 15 pmol of each primer (SEQ ID NO: 19 to 38), 50 ng of human genomic DNA ( Roche), and 50 μl of a reaction solution containing 1 U of KOD FX Neo. The reaction is performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 68 ° C., and 10 minutes. After completion of the reaction, 5 μl of the reaction solution is subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the approximately amplified DNA fragment is confirmed under ultraviolet irradiation.

The nucleic acid amplification method in the present invention may be a method of amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to the nucleic acid amplification reaction.

Purification is a method for separating contaminants such as tissues and cell walls of biological samples from DNA in biological samples. Methods for separating DNA using phenol or phenol / chloroform, ion exchange resins, glass filters, etc. Alternatively, there is a method of separating DNA with a reagent having a protein aggregation action. In the nucleic acid amplification method of the present invention, a biological sample may be added to a nucleic acid amplification reaction solution and amplified without taking these purification methods.

“A biological sample that has not undergone a purification process” refers to a biological sample itself, or a liquid biological sample diluted with a solvent such as water, or a solid biological sample added to a solvent such as water and crushed by heat. And the like.
When nucleic acids to be amplified such as organs and cells are present in the tissue of the sample, an act of destroying the tissue to extract the nucleic acid (destruction by physical treatment, destruction using a surfactant, etc.) Does not correspond to the purification referred to in the present invention. Further, the act of diluting the sample obtained by the above method or the biological sample with a buffer solution does not correspond to the purification referred to in the present invention.

The biological sample applied to the nucleic acid amplification method of the present invention is not particularly limited as long as it is a sample collected from a living body. For example, animal and plant tissues, body fluids, cells, bacteria, viruses and the like. Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1: 100 bp to 1 kb multiplex PCR
As a multiplex PCR evaluation method, comparison was performed by performing multiplex PCR using a target in the range of 100 to 1 kb.

PCR uses Buffer, dNTPs attached to KOD FX Neo (manufactured by Toyobo), enzyme solution (KOD FX Neo), 1 × PCR Buffer, and 15 pmol of each primer (SEQ ID NO: 1 to 18, Non-Patent Document 2) 50 μl of a reaction solution containing 50 ng of human genomic DNA (Roche) and 1 U of KOD FX Neo was prepared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 60 ° C., 30 seconds → 68 ° C., 1 minute.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of about amplified DNA fragment was confirmed under ultraviolet irradiation.

The results are shown in FIG. It was confirmed that KOD FX Neo showed the most uniform amplification.

Example 2: 1 kb to 10 kb multiplex PCR
As a multiplex PCR evaluation method, comparison was performed by performing multiplex PCR using a target in the range of 1 kb to 10 kb.

PCR was performed using Buffer, dNTPs, enzyme solution (KOD FX Neo) attached to KOD FX Neo (Toyobo), 1 × PCR Buffer, 15 pmol of each primer (SEQ ID NO: 19 to 36), 50 ng of human genomic DNA ( Roche) and 50 μl of a reaction solution containing 1 U of KOD FX Neo was prepared. The reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 68 ° C., 10 minutes.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of about amplified DNA fragment was confirmed under ultraviolet irradiation.

The results are shown in FIG. KOD polymerase was able to amplify the target of 10 kb, which was difficult with other polymerases, and the most uniform amplification was confirmed.

Example 3: 100 bp to 1 kb multiplex PCR from blood
Experiments were similarly performed by changing the multiplex PCR template of Example 1 from purified human genome to human blood.

PCR uses Buffer, dNTPs attached to KOD FX Neo (manufactured by Toyobo), enzyme solution (KOD FX Neo), 1 × PCR Buffer, and 15 pmol of each primer (SEQ ID NO: 1 to 18, Non-Patent Document 2) Then, 50 μl of a reaction solution containing 2.5 μl of human blood as a template and 1 U of KOD FX Neo was prepared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 60 ° C., 30 seconds → 68 ° C., 1 minute.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of about amplified DNA fragment was confirmed under ultraviolet irradiation.

The results are shown in FIG. In amplification from human blood, uniform amplification was confirmed for all targets.

Example 4: 1 kb to 10 kb multiplex PCR from blood
Experiments were conducted in the same manner except that the multiplex PCR template of Example 2 was changed from purified human genome to human blood.

PCR uses Buffer, dNTPs attached to KOD FX Neo (manufactured by Toyobo), enzyme solution (KOD FX Neo), 1 × PCR Buffer, and 15 pmol of each primer (SEQ ID NO: 19 to 36), 2.5 μl as template 50 μl of a reaction solution containing 1 human blood and 1 U KOD FX Neo was prepared. The reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 68 ° C., 10 minutes.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of about amplified DNA fragment was confirmed under ultraviolet irradiation.

  The results are shown in FIG. In amplification from human blood, uniform amplification was confirmed for all targets.

Example 5: Multiplex PCR with 10 kb × 10 target
Ten kinds of primer sets having an amplification length of 10 kb were amplified by multiplex PCR, and the amplification products were quantified by real-time PCR to compare biases.

PCR uses Buffer, dNTPs attached to KOD FX Neo (Toyobo), enzyme solution (KOD FX Neo), 1 × PCR Buffer, and 15 pmol primers (SEQ ID NOs: 35, 36, 57 to 74), template As a reaction solution, 50 μl of a reaction solution containing 50 ng of human genomic DNA (Roche) and 1 U of KOD FX Neo was prepared. The reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 98 cycles at 98 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 68 ° C., 10 minutes.

Next, in order to perform quantification, a calibration curve was prepared by real-time PCR. For real-time PCR, qPCR Mix attached to KOD SYBR qPCR Mix (manufactured by Toyobo) was used, and 1 × qPCR Mix, 50 × ROX and 4 pmol of each primer (SEQ ID NOs: 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56), and the standard curve dilution level is 4000 copies, 2000 copies, 1000 copies, 500 copies, 250 copies, 20 μl of reaction solutions were prepared using 125 copies, 62.5 copies of human genomic DNA (Roche) as a template. Using a real-time PCR device (Applied Biosystems 7500 Fast real-time PCR system), the reaction was repeated at 98 ° C. for 2 minutes, followed by 30 cycles of 98 ° C., 10 seconds → 60 ° C., 10 seconds → 68 ° C., 30 seconds. I went. After completion of the reaction, the target peak was confirmed by melting curve analysis, and a calibration curve was prepared.

The prepared calibration curve is shown in FIG.

Amplification product copy number was quantified to compare bias. Real-time PCR uses qPCR Mix attached to KOD SYBR qPCR Mix (manufactured by Toyobo), 1 × qPCR Mix, 50 × ROX and 15 pmol of each primer (SEQ ID NOs: 37 and 38, 39 and 40, 41 and 42, 43 and includes a 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56), 20μl of the reactions 10 ⁶ multiplex PCR product 1μl of 10 kb × 10 targets diluted as a template Each solution was prepared. Using a real-time PCR device (Applied Biosystems 7500 Fast real-time PCR system), the reaction was repeated at 98 ° C. for 2 minutes, followed by 30 cycles of 98 ° C., 10 seconds → 60 ° C., 10 seconds → 68 ° C., 30 seconds. I went. After completion of the reaction, a target peak appearing in the latter half of 80 ° C. was confirmed by melting curve analysis.

The number of copies is shown in FIG. In KOD FX Neo, the biased multiplex PCR product amount ratio was 4.58 times, and the bias was low.

The present invention makes it possible to carry out the multiplex PCR method more reliably and accurately, and is expected to greatly contribute to the fields of research and diagnosis in the bio field.

Claims (6)

A method of specifically amplifying two or more target regions with one reaction solution, wherein a biological sample that has not undergone a purification step is added to a nucleic acid amplification reaction, and the reaction solution contains a DNA polymerase derived from Thermococcus kodakaraensis , A method for amplifying a target nucleic acid in a biological sample, comprising at least one target region whose region is 1 kb or more.   The method according to claim 1, wherein the target area includes at least five target areas that are 1 kb or more.   The method according to claim 1, wherein the target area includes at least five target areas of 10 kb or more.   The method according to claim 1, wherein the target area is 10 or more areas. The method according to claim 1, wherein the biological sample is blood. The method according to any one of claims 1 to 5 , wherein the copy number bias of the amplification product is 100 times or less.