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WO2018203280A1 - Mélanges de nucléotides pour l'amplification et le séquençage de polymères d'acides nucléiques - Google Patents

Mélanges de nucléotides pour l'amplification et le séquençage de polymères d'acides nucléiques Download PDF

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Publication number
WO2018203280A1
WO2018203280A1 PCT/IB2018/053091 IB2018053091W WO2018203280A1 WO 2018203280 A1 WO2018203280 A1 WO 2018203280A1 IB 2018053091 W IB2018053091 W IB 2018053091W WO 2018203280 A1 WO2018203280 A1 WO 2018203280A1
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Prior art keywords
nucleotide
mixture
dntps
sequencing
amplification
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English (en)
Spanish (es)
Inventor
Carlos Jaime BARRIOS HERNÁNDEZ
Lola Xiomara BAUTISTA ROZO
Juan Antonio GONZÁLEZ BARRIOS
Daniel MARTÍNEZ FONG
Francisco José MARTÍNEZ PÉREZ
Enrique Mejía Ospino
Néstor Martin MUNIVE ARGÜELLES
Gabriel Rodrigo PEDRAZA FERREIRA
Silvia Daniela RAMÍREZ ARDILA
Wellman Antonio RIBÓN GÓMEZ
Refugio RODRÍGUEZ VÁZQUEZ
Stephen Tobe
Lina María VERA CALA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Toronto
UNIVERSIDAD INDUSTRIAL DE SANTANDER
Instituto De Seguridad Y Servicios Sociales De Los Trabajadores Del Estado
Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional
Original Assignee
University of Toronto
UNIVERSIDAD INDUSTRIAL DE SANTANDER
Instituto De Seguridad Y Servicios Sociales De Los Trabajadores Del Estado
Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional
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Publication of WO2018203280A1 publication Critical patent/WO2018203280A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention belongs to the area of biotechnology, biomedicine and basic science, particularly mixtures of nucleotides and / or their analogues in different proportions and / or molar concentration of each of them, to be used in amplification and sequencing processes of polymers of nucleic acids.
  • nucleic acid polymers and their sequence has been one of the fields of Biology that has revolutionized civilization. The success of this process is the result of several discoveries and achievements of biochemistry, of nucleic acid purification systems from biological or environmental sources and of the chemical synthesis of nucleic acids.
  • the essence of the reaction is at the level of its components, since any variant of nucleotide polymerization invariably requires, in addition to a suitable equipment, the nucleic acid segment from which the polymers will be obtained, a DNA or RNA polymerase to perform the binding of the respective nucleotides, a reaction buffer, one or two oligonucleotides to indicate the beginning and end of the polymer to be obtained and nucleotides or also called dNTPs that make up the polymer.
  • nucleotide polymerization reaction Even in the simplicity of the development and execution of the nucleotide polymerization reaction, in many cases, unwanted nucleotide polymers are obtained and / or do not correspond to the expected size. On other occasions, the nucleotide sequence obtained may have errors, generated by the same reaction, whereby the polymer obtained is not functional.
  • the previous problem is because the nature of each Nucleotide sequence is unique, therefore, following a common protocol for a set of nucleotide polymers of any origin is sometimes inappropriate, since the physicochemical characteristics of any of them will be determined by their concentration and nucleotide distribution.
  • nucleotide polymerization reaction Another way to carry out the nucleotide polymerization reaction is that in which each of the nucleotides and components are commercialized independently so that the user performs the mixture according to his need.
  • the DNA polymerase, its buffer and its cofactor are sold separately from the nucleotides, which are commercially available either individually, or in a single mixture, with equal (equimolar) molar concentrations of each one of them.
  • nucleotide mixtures with equimolar concentrations can be used to carry out nucleotide polymerization, it is often not possible to obtain complete and efficient polymerization, due to differences in nucleotide proportions of nucleotide polymers present in a biotechnological process and in each of the beings of nature.
  • the invention relates to mixtures of nucleotides (dNTPs), adenine, thymine, uracil, guanine and cytosine and / or their analogues, in different proportions and / or molar concentrations of each of them, to be used in the polymerization of nucleic acids.
  • dNTPs nucleotides
  • the invention also contemplates a process for the amplification and sequencing of nucleic acid polymers employing said mixtures.
  • FIG. 1 Comparison of polymers of the complementary DNA of Bos taurus bovine muscle beta and Gallus gallus bird when using Blend B and Blend D (lanes 2-5) with respect to a commercial mixture (lanes 6-9) .
  • FIG 2. Screening in the RACE type library for the identification of cDNAs related to the codon sequence for the FGLamide neuropeptide in the Flour Beetle. From lanes 1-8. M 100 bp marker (Invitrogen), Pl and P2 screening with the Oligonucleotides for FGLamide. C amplification controls. ES control without nucleotides.
  • FIG 3. Identification by PCR, using Mixture D, of some of the clones in recombinant plasmids whose inserts correspond to cDNAs related to the codons of the FGLamide of the brain and nervous system of the T. molitor meal beetle obtained from screening in the RACE Library indicated in FIG. 2. Recombinant plasmids generate amplicons larger than the polilinker (lane 5). Only clones larger than 400 bp were sequenced from this group.
  • FIG 6. Comparison of sequencing of nucleotide polymers with mixtures of nucleotides suggested by commercial house for genomes rich in GC (upper) and Mixture L with adjuvant (lower). DETAILED DESCRIPTION OF THE INVENTION
  • the invention relates to mixtures of nucleotides (dNTPs), adenine, thymine, uracil, guanine and cytosine (dNTPs) and / or their analogues, with different proportions and / or molar concentration (mol / mol) of each of them, which It provides a simple, fast and simple option to obtain and sequence nucleic acid polymers (eg DNA and RNA), even in nucleotide regions whose organization and physicochemical properties prevent their characterization with current methods and reagents.
  • dNTPs nucleotides
  • dNTPs adenine, thymine, uracil, guanine and cytosine
  • the term "and its analogs" refers to similar compounds of nitrogenous bases, which can be defined as nitrogenous bases with some modification or inclusion in their structure.
  • analogs there may be mentioned aminoalyl nucleotides, fluorophores, nucleotides bound to a thiol group or linked to a biotin and 2-aminopurine.
  • the proportion and / or molar concentration of the dNTPs in the mixtures of the present invention is established according to a bioinformatic model that allows analyzing the characteristics of the DNA or RNA polymers and their nucleotide content.
  • the molar concentration of each of the dNTPs can be between 5.0% and 45.0%.
  • Table 1 shows the ranges of each of the nucleotides.
  • the first value corresponds to the minimum and the second to the maximum of each nucleotide.
  • the molar ratio of Adenine-Thymine or Adenine-Uracil, like that of Guanine-Cytokine, is always 1: 1 and the total sum of the percentages of them is 100.0%.
  • the percentage of Adenine is 20, 1%
  • the percentage Timina is also 20, 1%, so, to complete 100%, the percentages of Guanine and Cytokine would have to be , each, of 29.9%.
  • the molar concentration of each of the dNTPs in the mixtures of the invention will be determined, in each case, by the nucleotide proportions of the nucleic acid polymers with which it must react.
  • the different molar concentration ranges of dNTPs in the mixtures of the invention allow them to be used to amplify and / or sequence nucleotide polymers with a dNTP content close to equality (eg 26, 1% AT and 23.9% GC ) or significantly different (eg 5.0% AT and 45.0 GC).
  • the mixtures of the invention may contain one or more adjuvants to favor the amplification and sequencing reactions of nucleic acid polymers.
  • adjuvants there may be mentioned solvents, diluents, salts, pH regulators, cofactors, enzymes and chemical compounds such as dimethylsulfoxide, ethylammonium tetrachloride (TAE-C1), methylammonium tetrachloride (TAM-C1), in concentrations between 0, 1% and 99.9% p / p.
  • mixtures of dNTPs may be accompanied by an acceptable carrier or vehicle, which should favor the stability and chemical properties of nucleotides.
  • acceptable vehicle for purposes of the present invention can be defined as a substance or mixture of substances (eg solvents, solutions) capable of containing dNTPs and / or adjuvants, without affecting their ability to perform the function. desired.
  • the determination of the nucleotide ratio of the polymer to be amplified or sequenced can be determined by the user according to his selection criteria.
  • Table 2 illustrates the nucleotide relationship of genomes, chromosomes and mitochondrial genomes in some bacteria, fungi, plants, animals and humans. With the result value you can select the most appropriate dNTP mix. Given the versatility of the mixtures of the invention, they can be used for polymerization reactions of deoxyribonucleic acid, ribonucleic acid, nucleotide polymer generation systems or their sequencing in all its variants.
  • the first value corresponds to the minimum and the second to the maximum of each nucleotide.
  • the mixtures of the invention (Mixtures B, D, E, L and W) have the percentages of dNTPs indicated in Tables 3 and 4.
  • the present invention also contemplates a process for the amplification and sequencing of nucleic acid polymers characterized in that it employs one or more mixtures of dNTPs indicated in Tables 1, 3 and 4.
  • the amplification and sequencing can be carried out by applying molecular techniques that include: hybridization, synthesis of complementary deoxyribonucleic acid (cDNA), nucleic acid amplification, nucleic acid sequencing, prediction by means of electronic, computer or manual technologies that allow determining the complementarity between nucleotide sequences and the concentration of their components in the polymer.
  • the process of the invention comprises the following steps: a) Purify the nucleotide polymers present in a sample of any origin;
  • c) Carry out a synthesis reaction of complementary nucleotide polymers and / or their amplification or sequencing under suitable conditions of reaction medium, pH and temperature. d) Establish whether the nucleotide polymer obtained corresponds to the size, composition and nucleotide order expected directly or indirectly, to be applied according to the user's selection.
  • the invention also contemplates the use of dNTP mixtures of the invention to amplify and sequence nucleic acid polymers by conventional and unconventional enzymatic methods.
  • EXAMPLE 1 Preparation of a mixture of different concentration of dNTPs.
  • EXAMPLE 2 Preparation of a mixture of different concentration of dNTPs with adjuvants for PCR. Similarly to the preparation described in Example 1, from stock solutions a mixture of dNTPs for one-step PCR will be prepared consisting of: 1 reaction buffer IX (20 mM Tris-HCl, 10 mM (NH4) 2S04, 10 mM KC1, 2 mM MgS04, 0.1% Triton X-100 pH 8.8 at 25 ° C), 2 Units of Deep Vent DNA polymerase (New England Biolabs 2 ®), 150 ⁇ of Adenine, 150 ⁇ of Thymine, 350 ⁇ of Guanine and 350 ⁇ of Cytokine and 5% of DMSO.
  • 1 reaction buffer IX (20 mM Tris-HCl, 10 mM (NH4) 2S04, 10 mM KC1, 2 mM MgS04, 0.1% Triton X-100 pH 8.8 at 25 ° C
  • 2 Units of Deep Vent DNA polymerase New
  • EXAMPLE 4 RT-PCR amplification of the complementary DNA segment immersed between the first and second universal domain of the bovine muscle beta actin Bos taurus and the Gallus gallus bird.
  • the obtaining and purification of total ribonucleic acid (RNA) from bovine and poultry muscle was carried out with the Trizol reagent according to the manufacturer's instructions (Invitrogen® 3 ).
  • the quantification and purity index was obtained by spectrophotometric standard methods, while the integrity of the total RNA was made by visualizing the ribosomal RNAs by their agarose gel staining.
  • the cDNA synthesis was performed with total RNA of each sample with the enzyme reverse transcriptase (SuperScript III Rt Invitrogen®) using a 21 thymine oligonucleotide and with equimolar nucleotide concentrations as polymerization initiator.
  • the reaction conditions, enzyme inactivation and removal of the tempered mRNA were carried out according to conventional enzymatic and physical methods.
  • Nucleotide polymer amplification was performed with a cofactor, a buffer, DNA polymerase (Taq Platinum Invitrogen®), a pair of sense and antisense oligonucleotides that hybridize approximately in the region of the first and second universal domain of beta actin (Joseph J., Fey P. et al. The actinome of Dictyostelium
  • EXAMPLE 5 Identification and characterization of cDNA polymers related to the precursor of the FGLamide nervous system of the Tenebrio molitor flour beetle.
  • RNA extraction was performed by standard tissue extraction techniques and then the extraction and purification of total RNA from all tissues was performed. Of the total total RNA set, only the 5'-end adapter oligonucleotide was attached to the messenger RNA by enzymatic methods.
  • This construction for this invention is called the RACE Library, which is amplified in two steps: 1) complementary DNA synthesis with a 3 'RACE oligonucleotide and 2) RT-PCR amplification with oligonucleotides for adapters.
  • screening in the RACE library is performed by an oligonucleotide for the nucleotide polymer to be found and another oligonucleotide for one of the adapters.
  • the products are cloned into plasmids and sequenced by standard methods to obtain the nucleotide polymer sequence.
  • the tissue was obtained using conventional dissection methods based on national and international biosafety and bioethics protocols for handling laboratory animals. From these tissues the total RNA was obtained analogously to that described in Example 4.
  • RNA oligonucleotide Prior to the synthesis of complementary DNA to obtain the RACE Library, the 5 'end of all messenger RNAs was modified by removing 7-methyl guanocin (CAP) and subsequently incorporating at this end an RNA oligonucleotide by enzymatic methods according to the Kit GeneRacer® 5 .
  • CAP 7-methyl guanocin
  • Complementary DNA synthesis was carried out with a 3 'RACE oligonucleotide in the same manner as in Example 4, but two mixtures of nucleotides were used: the first, a commercial mixture of Invitrogen® with equimolar concentrations of nucleotides , and the second, a mixture of nucleotides (Mixture E, Table 3) corresponding to the specific nucleotide composition for the Red Flour Beetle (TGSC 2008), which also included 5% dimethylsulfoxide (DMSO) as an adjuvant.
  • DMSO dimethylsulfoxide
  • the polymerization conditions were 28 cycles with a denaturation temperature of 95 ° C for 20 seconds, alignment of 62 ° C for 30 seconds, and extension at 72 ° C for 120 seconds.
  • the products were purified by column exclusion methods established by QIAGEN® and visualized on agarose gel.
  • the resulting nucleotide polymers were called the RACE Library.
  • the polymerization procedure indicated in the previous paragraph was performed, but using as a screening vehicle and polymerization initiator a degenerated oligonucleotide whose nucleotides correspond to the codons for the last FGLamide amino acids and the basic amino acid pair for propeptide processing (Mart ⁇ nez-Pérez F, Bendena WG, Chang BS, Tobe SS. FGLamide Allatostatin genes in Arthropoda: introns early or late? Peptides. 2009 Jul; 30 (7) : 1241-8; Ellott KL, Chan KK & Stay B.
  • the positive bacterial colonies were cultured independently in liquid medium with the selection drug and then the recombinant plasmids were purified by conventional chemical-enzymatic methods.
  • the selection of the positive plasmids of the false positives was carried out by PCR according to the amplification procedure of the RACE library, but with a pair of oligonucleotides flanking the polilinker region of each plasmid and for 20 cycles.
  • the amplicons were visualized on agarose gel as indicated in the screening RT-PCR reaction of the RACE library (FIG. 3).
  • FIG. 4 shows a couple of graphs of the luminosity pattern of the sequencing of three of the recombinant plasmids using the nucleotide mixture of the commercial product, where it is clearly observed that there was no sequencing reaction since there was no polymerization reaction.
  • sequencing the same recombinant plasmids for GC-rich nucleotide polymers generated amplicons without a biological agreement to any open messenger RNA reading frame, as shown in FIG. 5. Improper polymerization was corrected using Mixture E.
  • the result of the commercial mixture did not produce a sequence according to a biological pattern, as did the mixture E, in which the polymerization pattern is that expected for any nucleotide polymer regardless of its concentration and order of nucleotides (FIG. 6).
  • nucleotide polymers that could not be identified by the existing methods for the genomic sequencing of homologous species was confirmed (TGSC, 2008), but of which there were reports of the biological activity of the products proteins synthesized from the polymer corresponding nucleotide (Mart ⁇ nez-Pérez F, Bendena WG, Chang BS, Tobe SS.
  • EXAMPLE 6 Third generation genomic sequencing of the Kocuria rhizophila bacteria genome.
  • the bacterial species Kocuria rhizophila ATCC 9341 was selected, whose approximate nucleotide composition is AT 29.8% - GC 71, 2% (Takarada H, Sekine M, Kosugi H, Matsuo Y, Fujisawa T, Omata S, Kishi E, Shimizu A, Tsukatani N, Tanikawa S, Fujita N, Harayama S. Complete genome sequence of the soil actinomycete Kocuria rhizophila. J Bacteriol Jun 2008; 190 (12): 4139-46) and Mixture L was used (Table 3).
  • Genomic sequencing was performed with the Flx + 454-ROCHE 8 system in 1x550 format.
  • the Library was built with 1500 bp inserts and its titration was carried out according to the protocol proposed by the Rapid Library Preparation Manual GS FLX + and GS Junior + Series - XL 9 .
  • EXAMPLE 7 In vitro transcription of nucleotide polymers.
  • any commercial Kit containing the nucleotides (dNTPs) and an RNA polymerase can be used.
  • dNTPs nucleotides
  • MEGAscript® T7 Transcription kit 12 that includes the four nucleotides independently to obtain any of the mixtures mentioned in Examples 1 to 6.
  • a mixture of dNTPs is prepared with the proportions of the W Mix , as indicated in Table 4.
  • the RNA is purified and recovered following the indications of the product or by conventional chemical or physical methods.

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Abstract

La présente invention concerne des mélanges de nucléotides dNTP et/ou leurs analogues, selon différentes proportions et/ou concentrations molaires de chacun d'eux, qui permet une polymérisation optimale d'acides nucléiques indépendamment de leur teneur et distribution dans le polymère. L'invention concerne également un procédé pour l'amplification et le séquençage de polymères d'acides nucléiques caractérisé en ce qu'il utilise un ou plusieurs de ces mélanges de dNTP. Les mélanges peuvent être utilisés dans la production de polymères nucléotidiques dans n'importe laquelle des variantes de PCR, RT-PCR, ou dans le séquençage de ceux-ci par les procédés classiques, le pyroséquençage ou séquençage de troisième génération.
PCT/IB2018/053091 2017-05-03 2018-05-03 Mélanges de nucléotides pour l'amplification et le séquençage de polymères d'acides nucléiques Ceased WO2018203280A1 (fr)

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CONC2017/0004500 2017-05-03
CONC2017/0004500A CO2017004500A1 (es) 2017-05-03 2017-05-03 Mezclas de nucleótidos para la amplificación y secuenciación de polimeros de ácidos nucleícos

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5976842A (en) * 1997-10-30 1999-11-02 Clontech Laboratories, Inc. Methods and compositions for use in high fidelity polymerase chain reaction
US8409805B2 (en) * 2009-02-13 2013-04-02 Asuragen, Inc. Method of amplification of GC-rich DNA templates

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5976842A (en) * 1997-10-30 1999-11-02 Clontech Laboratories, Inc. Methods and compositions for use in high fidelity polymerase chain reaction
US8409805B2 (en) * 2009-02-13 2013-04-02 Asuragen, Inc. Method of amplification of GC-rich DNA templates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FROMANT, M. ET AL.: "Direct Random Mutagenesis of Gene -Sized DNA Fragments Using Polymerase Chain Reaction", ANALYTICAL BIOCHEMISTRY, vol. 224, no. 1, 1995, pages 347 - 353, XP000486749 *
VAN PELT-VERKUIL, E. ET AL.: "Deoxynucleotide Triphosphates and Buffer Components", PRINCIPLES AND TECHNICAL ASPECTS OF PCR AMPLIFICATION, 2008, pages 91 - 101, Retrieved from the Internet <URL:https://link.springer.com/chapter/10.1007/978-1-4020-6241-4_6> [retrieved on 20180414] *

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