JP6980998B2 - Nucleic acid amplification composition - Google Patents

Description

The present invention relates to the field of nucleic acid amplification. More specifically, the present invention relates to a composition for nucleic acid amplification for quantification of a library for a next-generation DNA sequencer, which is a large-scale sequence.

DNA sequence analysis is an important basic means for analyzing genetic information. In recent years, among DNA sequencers, next-generation sequencers that can obtain enormous amounts of DNA information in a short time compared to conventional capillary sequencers have emerged and are attracting attention. For example, a method of detecting the fluorescence intensity when resynthesizing one base at a time using a DNA fragment as a template and determining the base sequence can be mentioned. Next-generation sequencing is a technology that can simultaneously sequence thousands to millions of DNA molecules. Next-generation sequencing is revolutionizing fields such as individual medicine, hereditary diseases, and clinical diagnostics, as it enables advanced and high-speed processing such as the ability to sequence multiple individuals at the same time.

As described above, the great merit of the next-generation sequencer is that a huge amount of DNA information can be obtained in a short time, and it is applied to a wide range of fields from genetics and molecular biology to clinical medicine.
A huge amount of data can be obtained with the next-generation sequencer, but if the loading amount of the DNA library sample whose concentration is known in advance is not accurate, the quantity and quality of the obtained data will deteriorate, so the concentration of the library sample is quantified. It is important to find exactly.

As a method for quantifying the library concentration, there are spectrophotometric measurement (Nanodrop, etc.) and fluorescence measurement (Qubit, PicoGreen, etc.). However, in the case of Illumina's next-generation DNA sequencer, these measurement methods also measure DNA to which P5 and P7 tags bound on the flow cell are not added, so it is not possible to accurately determine the concentration of an effective library. There is a problem (Non-Patent Document 1).

On the other hand, real-time PCR quantification uses primers designed to anneal to the tag sequence added to the DNA library, so the concentration of an effective library that binds to the flow cell can be accurately quantified and is stable. You can get the data.

However, with Taq DNA polymerase, which is often used as a real-time PCR reagent, amplification becomes difficult when the amplification size of the library is long or the GC content is high, and accurate quantification is often impossible.

Scientific Reports Volume 6, pp. 1-10 (issued April 6, 2016)

The present invention has been made in the background of the problems of the prior art. That is, it is an object of the present invention to provide a nucleic acid amplification composition capable of accurately quantifying a target regardless of the type (size, GC content) of the DNA library.

As a result of diligent studies in view of the above circumstances, the present inventors have obtained a DNA polymerase having a long chain length and a DNA library having a high GC content, which has been difficult in the past, accurately by using a DNA polymerase belonging to Family B. We have found that it can be quantified and arrived at the present invention.

Item 1. A composition for quantifying a DNA library sample for a DNA sequencer having a GC content of 70% or more, which comprises a DNA polymerase belonging to Family B for nucleic acid amplification.
Item 2. A composition for quantifying a DNA library sample for a DNA sequencer having a size of 600 bp or more, which comprises a DNA polymerase belonging to Family B for nucleic acid amplification.
Item 3. Item 2. The composition for nucleic acid amplification according to Item 1 or 2, wherein the DNA polymerase belonging to the family B is derived from the genus Thermococcus or the genus Pyrococcus.
Item 4. Item 3. The composition for nucleic acid amplification according to Item 3, wherein the DNA polymerase belonging to the family B is derived from Thermococcus kodakaranesis.
Item 5. Item 3. The composition for nucleic acid amplification according to Item 3 or 4, wherein the DNA polymerase derived from the genus Thermococcus lacks 3'-5'exonuclease activity.

According to the present invention, the target can be accurately quantified regardless of the type of library (size, GC content).

It is a figure which shows the result of having examined the comparison by the size of the DNA library in Example 2. FIG. It is a figure which shows the result of having examined the comparison by the GC content of the DNA library in Example 4. FIG.

Hereinafter, the present invention will be described in detail.
One of the embodiments of the present invention is a composition for quantifying a DNA library sample for a DNA sequencer capable of amplifying a GC-rich target having a GC content of 70% or more, preferably 75% or more. It is a composition for nucleic acid amplification, which comprises a DNA polymerase belonging to Family B.

Further, as another embodiment of the present invention, a quantification of a DNA library sample for a DNA sequencer capable of amplifying a long-chain target having a size of 600 bp or more, preferably 700 bp or more, more preferably 800 bp or more. It is a composition for nucleic acid amplification, which comprises a DNA polymerase belonging to Family B.

In the present invention, the "DNA sequencer" means a device that reads and analyzes a base sequence of DNA. In particular, a device capable of high-speed processing of a huge amount of sequencing reactions is called a next-generation sequencer.

The "DNA library for a DNA sequencer" in the present invention means a collection of various DNA fragments prepared for a sequencer.

The DNA polymerase belonging to Family B in the present invention is preferably a DNA polymerase derived from archaea. Archaeal-derived DNA polymerases belong to either Pol I or Pol II of Family B.

In the present invention, it is preferably a DNA polymerase belonging to Pol I type. Examples of archaeal-derived DNA polymerases belonging to 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 Pryrococcus friosus and Pryrococcus sp. DNA polymerases isolated from GB-D, Pryrococos Woesis, Prococcus abysii, Prococcus horikoshii, but not limited to these. DNA polymerases derived from the genus Thermococcus include Thermococcus kodakaransis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9degree North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Includes, but is not limited to, DNA polymerases isolated from KS-1, Thermococcus celer, or Thermococcus sicili. These DNA polymerases are commercially available, Pfu (Starage), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Deep Vent (New England Biolabs), and so on.

In the present invention, the DNA polymerase belonging to Family B in the present invention preferably lacks 3'-5'exonuclease activity. Here, the 3'-5'exoase activity refers to the ability to remove the incorporated nucleotide from the 3'end of the DNA polymer.

A DNA polymerase that is "deficient in 3'-5'exonuclease activity" can consist of amino acid substitutions, deletions, or additions of the DNA polymerase. Modifications to the amino acids corresponding to positions 137-147, 206-222, and 308-318 in SEQ ID NO: 1 are shown. More preferably, it is a modification of at least one of the amino acids corresponding to positions 141, 142, 143, 147, 210 and 311 in SEQ ID NO: 1. More preferably, the amino acid modification is any one selected from D141A / E143A, I142R, N210D, or Y311F, a DNA polymerase lacking 3'-5'exonuclease activity.

Note that the (exo (-)) DNA polymerase lacking 3'-5'exonuclease activity includes a complete lack of activity, for example 0.03%, 0.05% compared to the parent enzyme. , 0.1%, 1%, 5%, 10%, 20%, or a modified DNA polymerase having an exonuclease activity of up to 50% or less. In the present invention, the method used to generate and analyze the 3'-5'exonuclease DNA polymerase shall be the method disclosed in US Pat. No. 6,946,273.

In the present invention, as the DNA polymerase, a mutant having been modified so as to have a reduced base analog detection activity may be used. The DNA polymerase belonging to Family B may be a mutant having reduced base analog detection activity. The base analog indicates a base other than adenine, cytosine, guanine, and thymine, and examples thereof include uracil and inosine. Normally, DNA polymerases belonging to Family B bind strongly when they detect base analogs such as uracil and inosine, and inhibit the function of DNA polymerase. The base analog detection activity indicates an activity that strongly binds to a base analog and inhibits the DNA polymerase function. The DNA polymerase variant belonging to Family B having reduced base analog detection activity is a DNA polymerase variant belonging to Family B characterized by a low binding ability to uracil and inosine. Such variants can be made by modifying at least one of the amino acid sequences for uracil binding (uracil binding pockets).

Specifically, among the amino acid sequences shown in SEQ ID NO: 1, a DNA polymerase having a sequence in which at least one of the amino acid residues at positions 7, 36 and 93 is replaced with another amino acid is used. Can be mentioned.

Specifically, the seventh tyrosine residue is preferably replaced with alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine. The 36th proline residue is preferably substituted with histidine, lysine or arginine. The 93rd valine residue is preferably replaced with lysine, hishidin or arginine.

The above-mentioned modified DNA polymerase can be obtained, for example, by the method disclosed in International Publication No. 2014/051031 pamphlet, but is not particularly limited.

Other configurations of the composition for nucleic acid amplification in the present invention are not particularly limited, but are a heat-resistant DNA polymerase, a nucleic acid as a template, one or more oligonucleotide primers, one or more deoxynucleotide triphosphates, or three deoxynucleotides. It preferably contains at least one of the group consisting of a derivative of phosphoric acid, a buffer, and a salt.

The one or more oligonucleotide primers used in the nucleic acid amplification composition of the present invention are oligonucleotides complementary to each nucleic acid strand of the nucleic acid to be amplified, and two or more kinds of primers are used. Is preferable. The primer is substantially complementary to each strand having a different sequence of double-stranded nucleic acids, and when the extension product synthesized from one primer is separated from the complement, the extension product is obtained. The other primer is selected so that it can serve as a template for the synthesis of extension products.

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

The nucleic acid amplification composition of the present invention preferably further contains a buffer. Examples of the buffer include Tris (TRIS), Tricine (TRICINE), Bis-Tricine (BIS-TRICINE), HEPES, MOPS, TES, TAPS, and PIPES. ), Caps (CAPS), etc., but are not particularly limited. The concentration of the buffer is preferably about 10 to 200 mM, more preferably about 20 to 100 mM. The pH is preferably in the range of 7.0 to 9.5, more preferably in the range of 7.5 to 9.0. Further, the buffer solution ^{preferably contains Mg 2+} at a concentration of 1 to 5 mM, and more preferably at a concentration of 1.5 to 2.5 mM. Further, KCl may be contained.

Further, the nucleic acid amplification composition of the present invention may contain albumin, glycerol, heparin, trehalose, betaine and the like. These addition ratios may be added within a range that does not inhibit the PCR reaction.

The nucleic acid amplification reaction solution in the present invention may contain a DNA detection agent in the nucleic acid amplification reaction solution for detection and monitoring of the amplification product. The DNA detector is not particularly limited. Examples thereof include a dye that binds to a phosphate group on the surface of DNA and a dye that intercalates between bases (intercalator), but it is preferable to use an intercalator. An intercalator refers to any molecule that binds to a nucleic acid in a reversible, non-covalent manner by inserting (intercalating) into double-stranded DNA, thereby indicating the presence and amount of nucleic acid. .. In general, an intercalator is a dye that is inserted into double-stranded DNA to fluoresce.

A large number of intercalators are known in the art and can also be used in the present invention. For example, Ethidium bromide, cyanine dyes (eg, TOTO®, YOYO®, BOBO and POPO), SYBR® Green I, SYBR® Green ER, SYBR® Green Gold, for example. , SYBR® DX, PicoGreen®, LCGreen®, EvaGreen®, SYSTEMX® Green, ResoLight, Propidium iodide, Acidine orange, 7-amino-actinomycin D, CyQUANT (registered trademark) GR, SYTO (registered trademark) 9, SYTO (registered trademark) 10, SYTO (registered trademark) 13, SYTO (registered trademark) 14, SYTO (registered trademark) 82, FUN-1 and the like. , Not particularly limited.

In the present invention, the nucleic acid amplification reaction may be carried out in the coexistence of a single-stranded binding protein, a double-stranded binding protein or a mismatch-binding protein.

Furthermore, an anti-DNA polymerase antibody having an activity of suppressing the polymerase activity and / or the 3'-5'exonuclease activity of the thermostable DNA polymerase may be used. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective in increasing the sensitivity of PCR and reducing non-specific amplification.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not particularly limited by the following examples.

Example 1: Preparation of DNA libraries of various sizes Library preparations with average fragment sizes of 400 bp, 600 bp, and 800 bp were performed. In order to obtain average fragment sizes of 400 bp, 600 bp and 800 bp, 100 ng of human genomic DNA (Roche) was used for 5 minutes (for 800 bp) and 7 minutes with the Kappa Frag Enzyme included in the kit using the Kapa Hyper Plus Kit (manufactured by Kappa Biosystems). The reaction was carried out with different treatment times (for 600 bp) and 10 minutes (for 400 bp). The treated product was electrophoresed on a 1% agarose gel, and the fragment-sized DNA to be used was extracted and purified from the gel by MagExtractor-PCR & Gel Clean Up- (manufactured by Toyobo). The library of purified DNA fragments was prepared according to the instruction manual.

The prepared library was quantified by Nanodrop (registered trademark), diluted with reference to the quantified value, and subjected to 10-fold dilution from 5.5 pg / μl as a template in 6 steps.

Example 2: Comparison by size of DNA library Using the prepared library as a template, the difference in PCR efficiency and quantitative value of each company depending on the size was compared. As a real-time PCR reagent, KOD SYBR (trademark registration) qPCR Mix (manufactured by Toyobo) using a KOD DNA polymerase from which 3'→ 5'exonuclease activity was removed was used. In addition, a DNA quantification standard for Illumina (manufactured by Kapa Biosystems) was used as a DNA standard for calculating quantified values. For real-time PCR, use the master mix attached to KOD SYBR (registered trademark) qPCR Mix, 1 × qPCR Mix, 1 × ROX, 4 pmol primers (SEQ ID NOs: 2 and 3), library template, or DNA quantification standard for Illumina. A 20 μl reaction solution containing the reaction solution was prepared. The reaction was carried out at 98 ° C. for 2 minutes, followed by a schedule of repeating 98 ° C. for 10 seconds → 65 ° C., 10 seconds → 68 ° C. for 1 minute for 35 cycles.

NEBNext (Trademark Registration) Library Quant Kit for Illumina (Trademark Registration) (manufactured by New England Biolabs) was used as the library quantification reagent to be compared. As a master mix, 100 μl of NEBNext (registered trademark) Library Quant Primer Mix was added to 1.5 mL of NEBNext (registered trademark) Library Quant Master Mix, and another 20 μl of ROX was added. A library template or a DNA quantification standard for Illumina was added to this mixed master mix to prepare 20 μl of reaction solution. The reaction was carried out on a schedule of repeating 35 cycles of 95 ° C., 15 seconds → 63 ° C., 45 seconds after the pre-reaction at 95 ° C. for 2 minutes.

The results of obtaining the PCR efficiency and the quantitative value from the obtained Ct value are shown in FIG. NEBNext (registered trademark) Library Quant Kit for Illumina (registered trademark) and Kappa Library Quantification Kit use KOD DNA polymerase from which 3'→ 5'exonuclease activity has been removed, whereas PCR efficiency decreases as the length increases. KOD SYBR® qPCR Mix had little effect on PCR efficiency due to length.

Example 3: Library preparation of various GC contents Library preparation with an average GC content of 50%, 70%, and 80% was carried out. Amplification was performed using 50 ng of human genomic DNA as a template using KOD FX Neo (manufactured by Toyobo) using primers (SEQ ID NOs: 4 to 21) to which Illumina P5 and P7 tags were added. The obtained PCR product was purified using MagExtractor-PCR & Gel Clean Up- (manufactured by Toyobo). Purified PCR products were combined to create libraries with GC contents of 50%, 70%, and 80%.

The prepared library was quantified by Nanodrop (registered trademark), diluted with reference to the quantified value, and subjected to 10-fold dilution from 5.5 pg / μl in 6 steps as a template.

Example 4: Comparison of DNA libraries by GC content Using the prepared DNA library as a template, differences in PCR efficiency depending on the GC content were compared. As a real-time PCR reagent, KOD SYBR ™ qPCR Mix, which uses a KOD DNA polymerase from which 3'→ 5'exonuclease activity has been removed, was used. In addition, the DNA quantification standard for Illumina was used as the DNA standard for calculating the quantification value. For real-time PCR, use the master mix attached to KOD SYBR (registered trademark) qPCR Mix, 1 × qPCR Mix, 1 × ROX, 4 pmol primers (SEQ ID NOs: 2 and 3), library template, or DNA quantification standard for Illumina. A 20 μl reaction solution containing the reaction solution was prepared. The reaction was carried out at 98 ° C. for 2 minutes, followed by a schedule of repeating 98 ° C. for 10 seconds → 65 ° C., 10 seconds → 68 ° C. for 1 minute for 35 cycles.

NEBNext (Trademark Registration) Library Quant Kit for Illumina (Trademark Registration) was used as the library quantification reagent to be compared. To 1.5 mL of NEBNext (registered trademark) Library Quant Master Mix as a master mix, 100 μl of NEBNext (registered trademark) Library Quant Primer Mix was added, and another 20 μl of ROX was added. A library template or a DNA quantification standard for Illumina was added to this mixed master mix to prepare 20 μl of a reaction solution. The reaction was carried out on a schedule of repeating 35 cycles of 95 ° C., 15 seconds → 63 ° C., 45 seconds after the pre-reaction at 95 ° C. for 2 minutes.

The result of obtaining the PCR efficiency from the obtained Ct value is shown in FIG. With NEBNext (Trademark Registration) Library Quant Kit for Illumina (Trademark Registration) and Kappa Library Quantification Kit, accurate PCR efficiency could not be calculated as the GC content increased. On the other hand, KOD SYBR (trademark registration) qPCR Mix using KOD DNA polymerase from which 3'→ 5'exonuclease activity was removed had little effect on PCR efficiency due to GC content.

Example 5: Next Generation-Cluster Density in Sequencer Using the above quantitative values, the next generation sequencer Miseq (registered trademark) manufactured by Illumina (registered trademark) is used in Miseq Regent Kit v2 (300 cycles) ( The run was performed using (manufactured by Illumina).

As a result of the run, a cluster density of 1076 k / mm ² was obtained, and it was proved that accurate quantification was possible by KOD SYBR (trademark registration) qPCR Mix.

According to the present invention, it can be accurately quantified regardless of the type (size, GC content) of the DNA library for a DNA sequencer.

Claims (5)

A composition for quantifying a DNA library sample for a DNA sequencer containing a nucleic acid having a GC content of 70% or more and a size of 600 to 800 bp, which comprises a DNA polymerase belonging to Family B. Nucleic acid amplification composition (except when it is a nucleic acid amplification reagent containing a DNA polymerase belonging to Family B, Proliferating Cell Nuclear Antique (PCNA) and betaine) . The composition for nucleic acid amplification according to claim 1, wherein the DNA polymerase belonging to the family B is derived from the genus Thermococcus or the genus Pyrococcus. The composition for nucleic acid amplification according to claim 1 or 2 , wherein the DNA polymerase belonging to the family B is derived from the genus Thermococcus. The composition for nucleic acid amplification according to any one of claims 1 to 3 , wherein the DNA polymerase belonging to the family B is derived from Thermococcus kodakaranesis. The composition for nucleic acid amplification according to any one of claims 1 to 4 , wherein the DNA polymerase belonging to the family B lacks 3'-5'exonuclease activity.

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