Nucleic acid amplification method

Abstract

The present application provides a high-sensitivity and high-yield nucleic acid amplification method using RNA. The nucleic acid amplification method includes a step of synthesizing cDNA from sample RNA through a reverse transcription reaction and a step of amplifying the cDNA using multiplex PCR, the reverse transcription and the multiplex PCR being performed in the presence of a single-stranded nucleic acid binding protein, which is T4 GP32 or a variant thereof.

Description

Technical Field

[0001] This invention relates to a method for nucleic acid amplification. Background Technology

[0002] Multiplex PCR is a method that amplifies multiple regions from a single DNA sample simultaneously using a PCR reaction with multiple primer pairs. Multiplex RT-PCR, which combines reverse transcription (RT) of RNA to cDNA with multiplex PCR, has been used for gene expression analysis and genotyping.

[0003] RNA is typically extracted from tissue samples. Furthermore, as a simpler and non-invasive method for RNA extraction, a method for extracting RNA from skin surface lipids (SSL) has recently been disclosed (Patent Document 1). The RNA contained in SSL is useful as an RNA sample for gene expression analysis, etc. However, the amount of SSL that can be recovered from a single subject is small, and the amount of RNA contained therein is also small; therefore, the amount of RNA that can be prepared using the SSL of a single subject is trace.

[0004] Non-patent literature 1 describes how BSA and T4 gene 32 protein (T4GP32) improve PCR reactions in the presence of inhibitors. Non-patent literature 2 describes how DMSO and betaine improve PCR specificity and yield. Non-patent literatures 3 and 4 describe how adding T4GP32 to the RT system significantly increases the yield of RT-PCR products, but no significant increase in yield is observed even when adding T4GP32 to the PCR system after RT. Patent literature 2 describes how, in the reverse transcription amplification method (RT-RamDA method) for amplifying cDNA using RNA as a template, T4GP32 binds to the single-stranded cDNA stripped from the template RNA, protecting the cDNA from nuclease degradation and thus increasing cDNA yield.

[0005] (Patent Document 1) International Publication No. 2018 / 008319

[0006] (Patent Document 2) International Publication No. 2016 / 052619

[0007] (Non-patent literature 1) Appl Environ Microbiol, 1996, 62(3): 1102-1106

[0008] (Non-patent literature 2) PLOS ONE, 2010, 5(6):e11024

[0009] (Non-patent literature 3) Appl Environ Microbiol, 1998, 64(2):669-677

[0010] (Non-patent literature 4) BioTechniques, 2001, 31(1): 81-86 Summary of the Invention

[0011] The present invention provides a nucleic acid amplification method, comprising: a step of synthesizing cDNA from sample RNA by reverse transcription; and a step of amplifying the cDNA by multiplex PCR, wherein the reverse transcription and the multiplex PCR are performed in the presence of a single-stranded nucleic acid binding protein, wherein the single-stranded nucleic acid binding protein is T4GP32 or a variant thereof.

[0012] The present invention also provides an improver, which is a yield improver for multiplex RT-PCR with a single-stranded nucleic acid binding protein as the active ingredient. The single-stranded nucleic acid binding protein is T4GP32 or a variant thereof, and is added to the reaction solution of both reverse transcription and multiplex PCR. Detailed Implementation

[0013] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference.

[0014] In this specification, "at least 90% identity" of an amino acid sequence or nucleotide sequence means 90% or more, preferably 95% or more, more preferably 97% or more, further preferably 98% or more, and even more preferably 99% or more.

[0015] In this specification, the identity of nucleotide and amino acid sequences can be calculated using the Lipman-Pearson method (Science, 1985, 227: 1435-41). Specifically, it can be calculated using the homology analysis program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software developer), with the Unit size to compare (ktup) set to 2.

[0016] Because there is a need to improve the sensitivity and yield of multiplex RT-PCR, this invention relates to a highly sensitive and high-yield method for nucleic acid amplification using RNA.

[0017] The inventors of this invention have discovered that by performing both the RT and PCR reactions in multiplex RT-PCR in the presence of T4GP32, high yields can be achieved even with trace amounts of RNA sample.

[0018] This invention improves the sensitivity and yield of multiplex RT-PCR. The method of this invention enables efficient amplification and analysis of minute RNA samples, such as RNA from SSL. This invention improves the sensitivity, accuracy, and efficiency of analyses using RNA samples (e.g., gene analysis, diagnostics, etc.).

[0019] This invention provides a method for nucleic acid amplification using RNA. The method includes the steps of synthesizing cDNA from sample RNA via reverse transcription and amplifying the cDNA using multiplex PCR.

[0020] The sample RNA used in the method of this invention can be any type of RNA, such as RNA from animals, plants, microorganisms, viruses, etc. The sample RNA can include mRNA, tRNA, rRNA, small RNA (e.g., microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), etc.), long intergenic non-coding (linc) RNA, etc. The sample RNA can contain one or more of the above-mentioned RNAs.

[0021] The RNA sample can be extracted from animals, plants, microorganisms, viruses, etc., using common methods. For example, the RNA can be extracted using the phenol-chloroform method, acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC), or variations thereof; methods using special magnetic particles coated with silica; methods using solid-phase reversible immobilization of magnetic particles; or commercially available RNA purification reagents (such as TRIzol Reagent, RNeasy, QIAzol, ISOGEN, etc.).

[0022] In a preferred embodiment, the sample RNA is RNA derived from skin surface lipids. In this specification, "skin surface lipids (SSL)" refers to lipid-soluble components present on the skin surface, sometimes also called sebum. Generally, SSL mainly contains secretions from exocrine glands such as sebaceous glands located in the skin, existing on the skin surface in the form of a thin layer covering the skin surface. SSL contains RNA from the skin cells of the subject, which can be used for biological analysis (Patent Document 1).

[0023] The subject from which RNA is extracted from the SSL can be any organism with the SSL on its skin. Examples of subjects include mammals, including humans and non-human mammals, with humans being preferred. For example, the subject can be a human or non-human mammal that needs to perform its own nucleic acid analysis. Alternatively, the subject can be a human or non-human mammal that needs or wants to perform gene expression analysis on its skin, or analyze the state of the skin or other parts of the body using nucleic acids.

[0024] The skin site from which SSL is collected from the tested subject can include any part of the body such as the head, face, neck, torso, hands, and feet; skin suffering from atopic inflammation, acne, xerosis, inflammation (redness), tumors, or other diseases; and skin with trauma, but there are no particular limitations. In this specification, unless otherwise specified, "skin" is a general term encompassing the epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands on the body surface.

[0025] The SSL of the test subject contains RNA expressed in the skin cells of the test subject, preferably RNA expressed in any part of the epidermis, sebaceous glands, hair follicles, sweat glands and dermis of the test subject, more preferably RNA expressed in any part of the epidermis, sebaceous glands, hair follicles and sweat glands of the test subject (see Patent Document 1). Therefore, the RNA from the SSL prepared by the method of the present invention is preferably RNA from at least one part of the epidermis, sebaceous glands, hair follicles, sweat glands and dermis of the test subject, more preferably RNA from at least one part of the epidermis, sebaceous glands, hair follicles and sweat glands.

[0026] When collecting SSL from the skin of a subject, any method for recovering or removing SSL from the skin can be used. SSL absorbent materials, SSL adhesive materials, or instruments for scraping SSL from the skin, as described later, are preferred. As for SSL absorbent or adhesive materials, there are no particular limitations as long as the material has an affinity for SSL; examples include polypropylene and pulp. More detailed examples of the process for collecting SSL from the skin include: methods using sheet materials such as blotting paper or blotting film to absorb SSL; methods using glass plates or tape to adhere SSL; and methods using scrapers or scrapers to scrape off and recover SSL. To improve SSL adsorption, SSL absorbent materials pre-containing highly lipid-soluble solvents can also be used. On the other hand, SSL absorbent materials containing highly water-soluble solvents or moisture will hinder SSL adsorption and are therefore not preferred. SSL absorbent materials are preferably used in a dry state.

[0027] The RNA-containing SSL collected from the sample can be preserved until the RNA preparation described later. The SSL can be preserved in its original state, absorbed or adhered to by an SSL absorbent or adhesive material. The SSL can be preserved under typical RNA preservation conditions (e.g., -80°C), but can be preserved under milder conditions (International Application No. PCT / JP2019 / 043040). For example, the temperature for preserving the RNA-containing SSL can be below 0°C, preferably below -10°C, more preferably below -20°C. The preservation time is not particularly limited, but is preferably below 12 months, more preferably below 6 months, and even more preferably below 3 months.

[0028] When extracting RNA from SSL, the usual RNA extraction methods described above can be used, such as the phenol-chloroform method, the AGPC method, and commercially available RNA purification reagents, chromatography columns, or magnetic particles.

[0029] In the method of this invention, cDNA is synthesized from the sample RNA by reverse transcription (RT), and then the cDNA is amplified by multiplex PCR (MPCR). Both the RT reaction and the MPCR are performed in the presence of single-stranded nucleic acid binding proteins.

[0030] Except for the addition of single-stranded nucleic acid-binding proteins to the reaction system, the reverse transcription of the sample RNA can be performed according to standard procedures. For example, simply culturing a reaction solution containing the sample RNA, primers, reverse transcriptase, and substrate allows the reverse transcriptase to act on the sample RNA and synthesize cDNA. As primers, primers targeting the specific RNA to be analyzed can be used. For more comprehensive nucleic acid preservation and analysis, oligomeric dT primers, random primers, or mixtures thereof are preferred. As reverse transcriptase, general reverse transcriptases can be used, preferably those with high accuracy and efficiency (e.g., M-MLV reverse transcriptase and its variants, or commercially available reverse transcription enzymes described later). The substrate is the substrate for the reverse transcriptase, preferably a deoxyribonucleotide, such as dATP, dCTP, dGTP, and dTTP, or mixtures thereof. The deoxyribonucleotide can also be modified. In this invention, commercially available reverse transcription enzymes and reagent kits can be used as enzymes and reagents for RT, such as the PrimeScript Reverse Transcriptase series (Takara Bio) and the SuperScript Reverse Transcriptase series (Life Technologies Japan Ltd.). SuperScript III Reverse Transcriptase and SuperScript VILO cDNA Synthesis kit (both from Life Technologies Japan Ltd.) are preferred. The temperature and time of the RT reaction can be appropriately set according to the type of enzyme or reagent used, the sample RNA, and the cDNA to be synthesized. For example, simply using a commercially available reverse transcription reagent, preparing the reaction solution according to its manual, and then adding a single-stranded nucleic acid binding protein to the reaction solution is sufficient. The RT conditions can also be set according to the reagent's manual.

[0031] The cDNA synthesized by RT is amplified using MPCR. Except for the addition of a single-stranded nucleic acid-binding protein to the reaction system, MPCR can be performed according to standard procedures. For example, PCR can be performed on a reaction solution containing cDNA, multiple primer pairs, a DNA synthase, and a substrate. The primer pairs can be appropriately designed based on the target DNA sequence. As the DNA synthase, a general DNA synthase can be used, preferably a DNA synthase with high amplification accuracy and efficiency (e.g., commercially available multiplex PCR enzymes described later). The substrate is the substrate of the DNA synthase, preferably a deoxyribonucleotide, such as dATP, dCTP, dGTP, and dTTP, or mixtures thereof. The deoxyribonucleotide can also be modified. In this invention, as the enzyme and other reagents used for MPCR, commercially available multiplex PCR enzymes and reagents can be used, such as the Ion AmpliSeq Transcriptome Human Gene Expression Kit (LifeTechnologies Japan Ltd.). The reaction temperature and time can be appropriately set according to the type of enzyme or reagent used, the template cDNA, etc. For example, you can simply use a commercially available multiplex PCR reagent, prepare the reaction solution according to its manual, and then add a single-stranded nucleic acid binding protein to the reaction solution. The PCR conditions can also be set according to the reagent's manual.

[0032] In the method of this invention, both the RT reaction and MPCR are performed in the presence of a single-stranded nucleic acid binding protein. Examples of such a single-stranded nucleic acid binding protein include T4GP32 (T4 gene32 protein) and its variants. T4GP32 is typically a protein consisting of the amino acid sequence of sequence number 1 and binds to single-stranded DNA. Variants of T4GP32 include proteins obtained by modifying or mutating any amino acid in the amino acid sequence of T4GP32. Examples of variants of T4GP32 include proteins consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of sequence number 1 and binds to single-stranded nucleic acid, preferably single-stranded DNA. T4GP32 is commercially available (e.g., through NEWENGLAND BioLabs Inc., Sigma-Aldrich, etc.). The initial concentration of the single-stranded nucleic acid binding protein in the reaction solution of the RT and MPCR is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 7 μg / mL or more. On the other hand, it is preferably 100 μg / mL or less, more preferably 20 μg / mL or less, and even more preferably 15 μg / mL or less. Alternatively, it is preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, and even more preferably 7 to 15 μg / mL.

[0033] In the method of this invention, RT and MPCR are preferably carried out in their respective reaction systems (two steps), but for simplicity, they can also be carried out in a single reaction system (one step). In a one-step reaction, the single-stranded nucleic acid binding protein used in RT can be directly used for MPCR.

[0034] Purification of the product obtained from this MPCR is preferably performed by size separation of the reaction product. Size separation allows the target PCR product to be separated from primers and other impurities contained in the MPCR reaction solution. DNA size separation can be performed using, for example, size separation columns, size separation chips, or magnetic beads suitable for size separation. A preferred example of magnetic beads suitable for size separation is Solid Phase Reversible Immobilization (SPRI) magnetic beads, such as Ampure XP. When Ampure XP and a DNA solution are mixed, DNA is adsorbed by carboxyl groups coated on the surface of the magnetic beads. The magnetic beads are then recovered using a magnet, thereby purifying the DNA. Furthermore, changing the mixing ratio of the Ampure XP solution to the DNA solution changes the molecular size of the DNA adsorbed on the magnetic beads. Using this principle, it is possible to recover DNA of a specific molecular size that is to be captured from the magnetic beads, while purifying DNA of other molecular sizes and impurities.

[0035] For purified PCR products, further processing may be necessary for subsequent analysis. For example, to perform DNA sequencing or fragment analysis, the purified PCR products can be prepared into appropriate buffer solutions, or the PCR primer regions in the amplified DNA can be cleaved, or adapter sequences can be added to the amplified DNA. For instance, the purified PCR products can be prepared into appropriate buffer solutions to remove PCR primer sequences and ligate adapters into the amplified DNA, allowing the resulting reaction products to be amplified as needed to create libraries for various analyses. These operations can be performed using the 5×VILO RT Reaction Mix included with the SuperScript (registered trademark) VILO cDNA Synthesis kit (Life Technologies Japan Ltd.), and the 5×Ion AmpliSeq HiFiMix and Ion AmpliSeq Transcriptome Human Gene Expression Core Panel included with the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Ltd.), following the operating procedures provided with each kit.

[0036] The nucleic acid amplification method of the present invention can be applied to various analyses or diagnoses using RNA. Preferably, the method of the present invention can be used for all analyses in which multiplex PCR can be applied, such as gene expression analysis, transcriptome analysis, functional analysis of the test subject, disease diagnosis, efficacy evaluation of drugs administered to the test subject, etc., for comprehensive gene analysis or expression analysis targeting specific genes.

[0037] As illustrative embodiments of the present invention, this specification further discloses the following substances, manufacturing methods, uses, and methods. However, the present invention is not limited to these embodiments.

[0038] [1] A method for nucleic acid amplification, comprising:

[0039] The step of synthesizing cDNA from sample RNA via reverse transcription; and

[0040] The step of amplifying this cDNA using multiplex PCR,

[0041] The reverse transcription and multiplex PCR were performed in the presence of a single-stranded nucleic acid binding protein, namely T4GP32 or a variant thereof.

[0042] [2] The method as described in [1], wherein preferably the T4GP32 or its variant is a protein consisting of the amino acid sequence of sequence number 1 or an amino acid sequence that is at least 90% identical to that sequence and is bound to a single-stranded nucleic acid.

[0043] [3] The method as described in [1] or [2], wherein the reverse transcription is preferably carried out in a reaction solution containing oligo-dT primers, random primers or mixtures thereof, more preferably in a reaction solution containing random primers.

[0044] [4] The method as described in any one of [1] to [3], wherein, preferably, the sample RNA is RNA derived from lipids on the skin surface.

[0045] [5] The method as described in any one of [1] to [4], wherein the initial concentration of the single-stranded nucleic acid binding protein in the reaction solution of the reverse transcription and the multiplex PCR is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 7 μg / mL or more. On the other hand, it is preferably 100 μg / mL or less, more preferably 20 μg / mL or less, and even more preferably 15 μg / mL or less, or preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, and even more preferably 7 to 15 μg / mL.

[0046] [6] An enhancer for multiplex RT-PCR yield enhancement, wherein the single-stranded nucleic acid binding protein is T4GP32 or a variant thereof, and is added to the reaction solution of both reverse transcription and multiplex PCR.

[0047] [7] The improver as described in [6], wherein preferably the T4GP32 or a variant thereof is a protein consisting of the amino acid sequence of sequence number 1 or an amino acid sequence that is at least 90% identical to that sequence and is bound to a single-stranded nucleic acid.

[0048] [8] The improver as described in [6] or [7], wherein the reverse transcription is preferably carried out in a reaction solution containing oligo-dT primers, random primers or mixtures thereof, more preferably in a reaction solution containing random primers.

[0049] [9] The improver as described in any one of [6] to [8] preferably improves the yield of multiplex RT-PCR using RNA derived from skin surface lipids.

[0050]

[10] The improver as described in any one of [6] to [9], wherein the initial concentration of the single-stranded nucleic acid binding protein in the reaction solution of the above-mentioned reverse transcription and multiplex PCR is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 7 μg / mL or more. On the other hand, it is preferably 100 μg / mL or less, more preferably 20 μg / mL or less, and even more preferably 15 μg / mL or less, or preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, and even more preferably 7 to 15 μg / mL.

[0051]

[11] The improver as described in any one of [6] to

[10] preferably improves the sensitivity and yield of multiplex RT-PCR.

[0052]

[12] Use of a single-stranded nucleic acid binding protein, T4GP32 or a variant thereof, in improving the yield of multiplex RT-PCR, and added to the reaction mixtures for both reverse transcription and multiplex PCR.

[0053]

[13] As described in

[12] , wherein preferably the T4GP32 or a variant thereof is a protein consisting of the amino acid sequence of sequence number 1 or an amino acid sequence that is at least 90% identical to that sequence and is bound to a single-stranded nucleic acid.

[0054]

[14] The use as described in

[12] or

[13] , wherein the reverse transcription is preferably carried out in a reaction solution containing oligo-dT primers, random primers or mixtures thereof, more preferably in a reaction solution containing random primers.

[0055]

[15] The use as described in any one of

[12] to

[14] , wherein the single-stranded nucleic acid binding protein is preferably used to improve the yield of multiplex RT-PCR using RNA derived from lipids on the skin surface.

[0056]

[16] The use as described in any one of

[12] to

[15] , wherein the initial concentration of the single-stranded nucleic acid binding protein in the reaction solution of the above-mentioned reverse transcription and multiplex PCR is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 7 μg / mL or more, on the other hand, preferably 100 μg / mL or less, more preferably 20 μg / mL or less, even more preferably 15 μg / mL or less, or preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, and even more preferably 7 to 15 μg / mL.

[0057]

[17] The use as described in any one of

[12] to

[16] , wherein the single-stranded nucleic acid binding protein described above is preferably used to improve the sensitivity and yield of multiplex RT-PCR.

[0058]

[18] Use of a single-stranded nucleic acid binding protein, which is T4GP32 or a variant thereof, in the production of a yield enhancer for multiplex RT-PCR, which is added to the reaction mixtures for both reverse transcription and multiplex PCR.

[0059]

[19] As described in

[18] , wherein preferably the T4GP32 or a variant thereof is a protein consisting of the amino acid sequence of sequence number 1 or an amino acid sequence that is at least 90% identical to that sequence and is bound to a single-stranded nucleic acid.

[0060]

[20] As described in

[18] or

[19] , the reverse transcription is preferably carried out in a reaction solution containing oligo-dT primers, random primers or mixtures thereof, more preferably in a reaction solution containing random primers.

[0061]

[21] The use as described in any one of

[18] to

[20] , wherein the single-stranded nucleic acid binding protein is preferably used to improve the yield of multiplex RT-PCR using RNA derived from lipids on the skin surface.

[0062]

[22] The use as described in any one of

[18] to

[21] , wherein the initial concentration of the single-stranded nucleic acid binding protein in the reaction solution of the reverse transcription and multiplex PCR is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 7 μg / mL or more, and on the other hand, preferably 100 μg / mL or less, more preferably 20 μg / mL or less, even more preferably 15 μg / mL or less, or preferably 1 to 100 μg / mL, more preferably 5 to 20 μg / mL, and even more preferably 7 to 15 μg / mL.

[0063]

[23] The use as described in any one of

[18] to

[22] , wherein the single-stranded nucleic acid binding protein described above is preferably used to improve the sensitivity and yield of multiplex RT-PCR.

[0064] Example

[0065] The present invention will now be described in more detail based on embodiments, but the invention is not limited thereto.

[0066] Example 1: Effect of T4GP32 on multiplex PCR

[0067] 1) Extracting RNA from SSL

[0068] One healthy adult female and one healthy adult male were selected as subjects. Sebum was collected from the entire face of the subjects using an oil-absorbing membrane (5×8cm, polypropylene, 3M Japan). The sebum-containing membrane was collected and transferred to a glass bottle, stored at -80°C until RNA purification. The RNA in the SSL contained in the oil-absorbing membrane was purified. The membrane was cut into appropriate sizes, and the aqueous phase containing RNA was recovered from the membrane using QIAzol Lysis Reagent (QIAGEN). RNA was extracted using the recovered aqueous phase according to the operating procedures provided with RNeasy (QIAGEN).

[0069] 2) Reverse transcription and multiplex PCR

[0070] Using the extracted RNA, multiplex PCR was performed using the SuperScript (registered trademark) VILO cDNA Synthesis kit (LifeTechnologies Japan Ltd.) with PCR enhancers under the following conditions: [99°C, 2 min → (99°C, 15 s → 60°C, 16 min) × 20 cycles → 4°C, Hold]. As PCR enhancers, based on existing reports (Non-Patent Literature 1, 2), BSA (lyophilized powder, essentially IgG-free, low endotoxin, BioReagent, suitable for cell culture) (Sigma Aldrich, Catalog No.: A2058), betaine hydrochloride (Fujifilm and Wako Pure Chemical Industries, Ltd.), and T4GP32 (NEW ENGLAND BioLabs Inc.) were used at the concentrations described in Table 1. The concentration of PCR products in the prepared library was determined using a Tapestation (Agilent Technologies, Inc.) and a High Sensitivity D1000 ScreenTape (Agilent Technologies, Inc.). The relative yield of the PCR products compared to the control (without PCR enhancer) was calculated.

[0071] 3) Results

[0072] The relative yields of PCR products in the presence of the PCR enhancer are shown in Table 1. In Table 1, the results for BSA and betaine are the results when using RNA from SSL obtained from female subjects, and the results for T4GP32 are the results when using RNA from SSL obtained from male subjects. With the addition of T4GP32, the relative yield of PCR products compared to the control increased to 1.41. On the other hand, with the addition of BSA or betaine, PCR products were detected in the control, while the PCR product yield decreased to below the detection limit. Prior art (Non-Patent Literature 3, 4) has reported that no significant increase in yield was observed even when adding T4GP32 to the PCR system after RT; however, this invention hypothesizes that in the case of multiplex PCR targeting multiple regions as described in this invention, the addition of T4GP32 has a significant effect on reaction sensitivity and yield.

[0073] [Table 1]

[0074]

[0075] Example 2: Effects of T4GP32 on reverse transcription and multiplex PCR

[0076] 1) Reverse transcription and multiplex PCR

[0077] One healthy adult male was selected as the subject. Sebum was collected from the subject's entire face using an oil-absorbing membrane (5×8cm, polypropylene, 3M Japan). RNA was extracted using the same procedure as in Example 1. Reverse transcription was performed as in Example 1, with or without T4GP32 (0.0010 w / v%). Multiplex PCR was also performed as in Example 1, with or without T4GP32 (0.0010 w / v%), and the PCR product concentration was determined. The relative yield of the PCR product compared to the control (without T4GP32 in both reverse transcription and PCR) was calculated.

[0078] 2) Results

[0079] The results are shown in Table 2. The relative yields compared to the control were 1.06 and 1.41 when T4GP32 was added only in reverse transcription and only in multiplex PCR, respectively. In contrast, the relative yield increased significantly to 2.19 when T4GP32 was added in both reverse transcription and multiplex PCR.

[0080] [Table 2]

[0081] sequence list <110> Kao Co., Ltd. <120> Nucleic acid amplification methods <130> KS1720 <150> JP 2020-002506 <151> 2020-01-10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 301 <212> PRT <213> Bacteriophage T4 <400> 1 Met Phe Lys Arg Lys Ser Thr Ala Glu Leu Ala Ala Gln Met Ala Lys 1 5 10 15 Leu Asn Gly Asn Lys Gly Phe Ser Ser Glu Asp Lys Gly Glu Trp Lys 20 25 30 Leu Lys Leu Asp Asn Ala Gly Asn Gly Gln Ala Val Ile Arg Phe Leu 35 40 45 Pro Ser Lys Asn Asp Glu Gln Ala Pro Phe Ala Ile Leu Val Asn His 50 55 60 Gly Phe Lys Lys Asn Gly Lys Trp Tyr Ile Glu Thr Cys Ser Ser Thr 65 70 75 80 His Gly Asp Tyr Asp Ser Cys Pro Val Cys Gln Tyr Ile Ser Lys Asn 85 90 95 Asp Leu Tyr Asn Thr Asp Asn Lys Glu Tyr Ser Leu Val Lys Arg Lys 100 105 110 Thr Ser Tyr Trp Ala Asn Ile Leu Val Val Lys Asp Pro Ala Ala Pro 115 120 125 Glu Asn Glu Gly Lys Val Phe Lys Tyr Arg Phe Gly Lys Lys Ile Trp 130 135 140 Asp Lys Ile Asn Ala Met Ile Ala Val Asp Val Glu Met Gly Glu Thr 145 150 155 160 Pro Val Asp Val Thr Cys Pro Trp Glu Gly Ala Asn Phe Val Leu Lys 165 170 175 Val Lys Gln Val Ser Gly Phe Ser Asn Tyr Asp Glu Ser Lys Phe Leu 180 185 190 Asn Gln Ser Ala Ile Pro Asn Ile Asp Asp Glu Ser Phe Gln Lys Glu 195 200 205 Leu Phe Glu Gln Met Val Asp Leu Ser Glu Met Thr Ser Lys Asp Lys 210 215 220 Phe Lys Ser Phe Glu Glu Leu Asn Thr Lys Phe Gly Gln Val Met Gly 225 230 235 240 Thr Ala Val Met Gly Gly Ala Ala Ala Thr Ala Ala Lys Lys Ala Asp 245 250 255 Lys Val Ala Asp Asp Leu Asp Ala Phe Asn Val Asp Asp Phe Asn Thr 260 265 270 Lys Thr Glu Asp Asp Phe Met Ser Ser Ser Ser Gly Ser Ser Ser Ser 275 280 285 Ala Asp Asp Thr Asp Leu Asp Asp Leu Leu Asn Asp Leu 290 295 300

Claims

1. A method for nucleic acid amplification, characterized in that, include: The step of synthesizing cDNA from sample RNA derived from lipids on the skin surface via reverse transcription; and The step of amplifying this cDNA using multiplex PCR, The reverse transcription and multiplex PCR were performed in the presence of a single-stranded nucleic acid-binding protein, T4GP32. The initial concentration of the single-stranded nucleic acid binding protein in the reaction solution of the reverse transcription and the multiplex PCR is 7–15 μg / mL. The reverse transcription and the multiplex PCR are performed in their respective reaction systems.

2. The method as described in claim 1, characterized in that: The T4GP32 is a protein consisting of the amino acid sequence of sequence number 1 and bound to a single-stranded nucleic acid.

3. The method as described in claim 1 or 2, characterized in that: The reverse transcription reaction is carried out in a reaction solution containing random primers.

4. The use of single-stranded nucleic acid binding proteins in improving the yield of multiplex RT-PCR, characterized by: This multiplex RT-PCR uses sample RNA derived from lipids on the skin surface. The single-stranded nucleic acid binding protein is T4GP32, and it was added to the reaction solutions of both reverse transcription and multiplex PCR at an initial concentration of 7–15 μg / mL. The reverse transcription and the multiplex PCR are performed in their respective reaction systems.

5. The use as described in claim 4, characterized in that: The T4GP32 is a protein consisting of the amino acid sequence of sequence number 1 and bound to a single-stranded nucleic acid.

6. The use as described in claim 4 or 5, characterized in that: The single-stranded nucleic acid binding protein is used to improve the sensitivity and yield of multiplex RT-PCR.

7. The use of single-stranded nucleic acid binding proteins in the production of yield improvers for multiplex RT-PCR, characterized in that: This multiplex RT-PCR uses sample RNA derived from lipids on the skin surface. The single-stranded nucleic acid binding protein is T4GP32. This modifier is added to the reaction solutions of both reverse transcription and multiplex PCR at an initial concentration of 7–15 μg / mL. The reverse transcription and the multiplex PCR are performed in their respective reaction systems.

8. The use as described in claim 7, characterized in that: The T4GP32 is a protein consisting of the amino acid sequence of sequence number 1 and bound to a single-stranded nucleic acid.

9. The use as described in claim 7 or 8, characterized in that: The single-stranded nucleic acid binding protein is used to improve the sensitivity and yield of multiplex RT-PCR.

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