KR102200109B1 - Nucleotide for amplification of target nucleic acid sequence and amplification mathod usning the same - Google Patents

Abstract

The present invention relates to an oligonucleotide for amplification of a target sequence and a method for amplifying a target sequence using the same, and more particularly, including crisper-cas9 or a system corresponding thereto, in a target sequence through reaction with a CAS 9 enzyme. It relates to an oligonucleotide that detects a position capable of binding and is amplified by binding to a target sequence at room temperature without special equipment, and a method of amplifying a target sequence using the same.

Description

Oligonucleotide for amplifying a target sequence, and a method of amplifying a target sequence using the same {NUCLEOTIDE FOR AMPLIFICATION OF TARGET NUCLEIC ACID SEQUENCE AND AMPLIFICATION MATHOD USNING THE SAME}

The present invention relates to an oligonucleotide for amplification of a target sequence and a method for amplifying a target sequence using the same, and more particularly, including crisper-cas9 or a system corresponding thereto, in a target sequence through reaction with a CAS 9 enzyme. It relates to an oligonucleotide that detects a position capable of binding and is amplified by binding to a target sequence at room temperature without special equipment, and a method of amplifying a target sequence using the same.

Isothermal amplification is a technology that performs a DNA amplification reaction at the same temperature, and various amplification methods such as LAMP (Loop-mediated isothermal amplification), RCA (Rolling circle amplification), and SDA (Strand displacement amplification) are being developed.

This isothermal amplification technology is getting more attention with the expansion of the on-site diagnosis market, and new products are emerging by being grafted into various field diagnosis devices. However, there are still many entry barriers for the on-site diagnosis technology to be performed by ordinary people without specialized skills.

The recently developed RNA-guided CRISPR (clustered regularly interspaced short palindromerepeats)-associated nuclease Cas9-based genome editing provides targeted knock-out, transcriptional activation and single guide RNA (sgRNA). (I.e., crRNA-tracrRNA fusion transcript) provides a breakthrough technology for inhibition, and this technology has demonstrated scalability by targeting numerous gene locations.

The short palindromic repeats (CRISPR) and CRISPR-related (Cas) systems, distributed at periodic intervals in cluster form, are the prokaryotic immune system first discovered by Ishino in E. coli (Ishino et al. 1987 (Journal of Bacteriology 169 (12): 5429-5433 (1987)). "Cas9 endonucleases" are a large group of endonucleases that catalyze double-stranded DNA cleavage in the CRISPR Cas system. This immune system provides immunity against viruses and plasmids by targeting the nucleic acids of viruses and plasmids in a sequence specific manner.

The CRISPR/Cas system incorporates endonucleases, meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), which may require manipulation of new proteins for all new target loci. It is known to be superior to other accompanying genome editing methods. There are several different CRISPR/Cas systems, and their nomenclature and classification changed as the system was further characterized. In the type II system, there are two strands of RNA that are part of the CRISPR/Cas system, namely CRISPR RNA (crRNA) and transcriptionally activated CRISPR RNA (tracrRNA). The tracrRNA hybridizes to the complementary region of the pre-crRNA to induce maturation of the pre-crRNA to the crRNA. The duplex formed by tracrRNA and crRNA is recognized by and associated with Cas9, a protein that is directed to the target nucleic acid by the sequence of the crRNA that is complementary to and hybridizes to the sequence in the target nucleic acid. It has been demonstrated that this minimal component of the RNA-based immune system can be reprogrammed into target DNA in a site-specific manner using a single protein and two RNA guide sequences or a single RNA molecule.

In another study, an argonate protein (NgAgo) isolated from the species of Natronobacterium gregoryi was reported (. Gao F, Shen XZ, Jiang F, Wu Y, Han C. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat. Biotechnol 2016;34:768-73), which has a gene editing function similar to that of existing Cas9, but it does not use RNA and uses single-stranded DNA as a guide. These features suggest a new alternative that can utilize the gene editing function using DNA primers not only in cells but also in in-vitro experiments.

However, most of these CRISPR/Cas systems are used as functions for gene editing, and have not yet been used in the field of diagnosis.

In the present invention, by applying the function of CRISPER-CAS9 or the corresponding gene editing system to provide an oligonucleotide for amplifying a target sequence capable of performing isothermal amplification at room temperature without special equipment, and a method of amplifying a target sequence using the same The purpose.

The present invention relates to an oligonucleotide for amplifying a target DNA sequence in order to solve the above problems, and an oligonucleotide for amplifying a target DNA sequence that hybridizes to a target DNA sequence and reacts with Cas 9 enzyme (Catalytically Cas 9) Provides.

Figure 2 shows the amplification process using the oligonucleotide for DNA amplification according to the present invention. In the amplification process using the oligonucleotide for DNA amplification according to the present invention, the oligonucleotide for DNA amplification according to the present invention hybridizes to the target DNA sequence by an enzyme capable of operating Crispr-cas9 as a Cas 9 enzyme. Cas 9) By reacting and binding with the enzyme, unlike the general Crispr-cas9 process shown in FIG. 1, it is possible to perform the amplification process even if a separate tracerRNA is not bound.

In the oligonucleotide for DNA amplification according to the present invention, the oligonucleotide for DNA amplification is 5'phosphorylated.

In the oligonucleotide for amplification of the target DNA sequence according to the present invention, the Cas 9 enzyme reacted with the oligonucleotide for DNA amplification (Catalytically Cas 9) is characterized in that the endonuclease function is lost. The oligonucleotide for amplification of the target DNA sequence according to the present invention has the main purpose of amplifying the target sequence, and it is preferable that the Cas 9 enzyme (Catalytically Cas 9) cuts the gene and loses the endonuclease function of inserting a specific gene. .

In the oligonucleotide for amplifying a target DNA sequence according to the present invention, the oligonucleotide for amplifying DNA is characterized in that it is a DNA sequence.

In the oligonucleotide for amplification of the target DNA sequence according to the present invention, the Cas 9 enzyme reacted by the DNA amplification oligonucleotide (Catalytically Cas 9) is an enzyme capable of operating as a Cas 9 enzyme when the guide sequence is DNA. Not specifically, Natronobacteria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Sphaerochaeta, Azospirillum, Gluconacetobacterium, Neissculeria, Nitraphylla, and Staphyloccus, Mycoplasma, and Staphyburia, Mycoplasma and Roseburia, It is possible to use those derived from the genus of microorganisms selected from the group consisting of Campylobacter. Among them, it has been reported that the CAS 9 enzyme derived from Natronobacteria functions as a CAS 9 enzyme even when the guide sequence is DNA (DNA-guided genome editing using the Natronobacterium gregoryi Argonaute, Nature Biotechnology volume 34, pages 768-773. (2016)).

The present invention also relates to an oligonucleotide for amplifying a target DNA sequence, comprising: an oligonucleotide for amplifying the DNA; And a stem sequence linked to the oligonucleotide. And a tracrRNA segment comprising a nucleotide sequence partially or completely complementary to the stem sequence; It provides a second oligonucleotide for DNA amplification comprising a.

The oligonucleotide for DNA amplification according to the present invention reacts with Cas 9 enzyme (Catalytically Cas 9) and contains only an oligonucleotide sequence complementary to the target sequence by reaction with Cas 9 enzyme due to the characteristics of Cas 9 enzyme. Amplification is possible, but stem sequence linked to the oligonucleotide; And a tracrRNA segment comprising a nucleotide sequence partially or completely complementary to the stem sequence; It is possible to further include.

The oligonucleotide for amplification of the target DNA sequence according to the present invention can be designed in various ways that are obvious to those skilled in the art, and mutations in the gene to be amplified in the gene to be amplified when amplifying the gene using the result of multiple sequence alignment and Shannon entropy are It is possible to design by selecting the smallest area.

More specifically, a first step of performing multiple sequence alignment of a plurality of gene data;

A second step of calculating a first column entropy for each column from the multi-sequence aligned data in the first step;

Forward and reverse candidate primers, and forward and reverse primers while changing the length while moving the starting position from the sequence of the entire gene aligned in the second step to the last nucleotide sequence A third step of generating a primer set of;

For each of the candidate primers generated in the third step, a position-specific gradient penalty value reflecting the position specificity in the primer is applied to the first column entropy value calculated in the second step. A fourth step of calculating a second column entropy;

The agent for calculating the primer entropy by summing the second column entropy in all sequences in the primer for the candidate primer set of the forward and reverse primers generated in the third step. Step 5;

A sixth step of calculating a primer set matching score of the forward and reverse primers by reflecting at least one of a primer entropy value of the fifth step, a primer length, a GC fraction, a Tm temperature, a hairpin, and a dimer score; And

It is possible to design including a seventh step of selecting forward and reverse primers based on the primer set matching score of the sixth step.

In this way, a primer entropy obtained by summing the first column entropy using the multi-sequence alignment result and the Shannon entropy, and the second column entropy reflecting the gradient penalty reflecting the position-specific conditions of each sequence in the primer Based on the used primer set matching score, it is possible to select an oligonucleotide for amplification of the target DNA sequence as a primer by selecting a more complementary region of the gene sequence.

Multiple sequence alignment is to align three or more nucleic acid or protein sequences, and a representative algorithm of multiple sequence alignment is a progressive alignment method, and a representative tool is ClustalW.

In the present invention, multiple sequence alignment was performed using ClustalW. First, start the alignment from the two most similar sequences, create a distance matrix/distance function for each aligned sequence pair, and create a phylogenetic guide from the matrice at the last node of the sequence. Create a tree. Then, when aligning the next new sequence to the aligned sequence, it is aligned using a guide tree. Going back to the first step, multiple sequence alignments are completed in complete order by continuing to add sequences to the multiple sequence alignment.

The term'first column entropy' of the present invention refers to a value obtained by calculating the degree of mutation in each column of a multi-sequence aligned file of genetic data using Shannon entropy. do. In the method for designing a primer for selectively amplifying a target gene according to the present invention, in the second step, a first column entropy value is calculated according to Equation 1 below.

[Equation 1]

(In Equation 1, Pa is the gene mutation fraction at the corresponding position, K is the column height by the number of samples subjected to multi-sequence treatment, i.e., the number of species, i represents the specific position of the gene, and entropy (i) is the multi-sequence alignment data. Represents the column entropy value at the i position of)

In an embodiment of the present invention, a search for a region having the least genetic variation in an alignment file obtained through multiple sequence alignment was performed using the feature of increasing entropy in a genomic sequence region where mutations are frequent. Specifically, the result of the multi-sequence alignment was converted into a matrix array, and the first column entropy was calculated by applying Shannon entropy for each column of the matrix. In an embodiment of the present invention, it is possible to select a filtering condition for a conserved fraction so that low-frequency genetic variation can be ignored when designing a primer. For example, reports of genetic variation of less than 5% can be excluded because the incidence of genetic variation is not large in nature.

In the present invention, the length of the primer is changed to a length of 15 to 25 bp while moving the starting position of the primer at 1 bp intervals from the first nucleotide sequence to the last nucleotide sequence. In the meantime, candidate primers were generated, and a set of forward primers and reverse primers in which these primers were combined were generated.

In the present invention, first, a candidate primer and a candidate primer set are generated by varying the position and length as described above, and in the fourth step, a gradient penalty in which the position-specific condition in the candidate primer is reflected in the first column entropy ( gradient penalty) is applied to calculate the second column entropy, and in the fifth step, the second column position entropy values for all gene position sequences in the candidate primer set are summed to calculate the primer entropy. ).

In the present invention, the corrected first column entropy value calculated by applying a gradient penalty to the first column entropy is referred to as a second column entropy. In the present invention, in order to correct the position-specific effect on the entropy reduction effect, which occurs when the primer length is generally short in the process of applying the Shannon entropy, and the remarkable entropy increase effect that appears at the 3'end of the primer sequence, the first The second column entropy was calculated by applying a gradient penalty value to the column entropy value.

Even in the case of primers starting at the same position, the shorter primer length will have the longer primer length or lower entropy. In the present invention, a gradient penalty was applied to correct this phenomenon and give importance to the genetic variation at a position close to the 3'end of the primer sequence. That is, the present invention is a gradient penalty value to the first column entropy value in order to reflect the primer binding decrease phenomenon generally appearing toward the 3'end of the primer sequence in the process of applying the Sannon entropy to the entropy. The entropy of the second column was calculated by applying.

That is, in the method of designing a primer for selectively amplifying a target gene according to the present invention, the first column entropy quantifies the degree of variation appearing in a plurality of gene data, and the second column entropy is within the primer for each sequence within the primer. The influence of the site-specific conditions in was quantified.

In the primer design method for detecting the target gene of the present invention, in the fourth step, a gradient penalty value η (i) at a specific position i in the primer is calculated according to Equation 2 below. .

[Equation 2]

(In Equation 2, λ _w is the weight of the gradient penalty, l _p is the primer length, D _i is the distance from the 5'end of the primer to the specific sequence position i, and τ(i) is the specificity of the primer. Is the number of the type of genetic mutation at sequence position i.)

At this time, the weight (λ _w ) of the gradient penalty may be defined by the user, has a value of 0 or more, and the initial value is set to 1.

In the primer design method for detecting the target gene of the present invention, in the fourth step, the second column entropy value for each row for all positions in the candidate primer is calculated according to Equation 3 below. To do.

[Equation 3]

(In Equation 3, η(i) is a gradient penalty at a specific position i according to Equation 2, and entropy(i) is a column entropy at a specific position according to Equation 1.)

In the primer design method for detecting the target gene of the present invention, the primer entropy value in the fifth step is the following formula using the second column entropy Q(i) according to Equation 3 above. It is characterized in that it is calculated according to 4.

[Equation 4]

(In Equation 4, K is the primer length, and Q(i) is the second column entropy at a specific position i according to Equation 3)

In the primer design method for detecting the target gene of the present invention, in the sixth step, based on a primer entropy value calculated in the fifth step for each candidate primer generated in the third step. It is characterized by calculating a pair matching score for each primer set according to Equation 5 below for a candidate primer set combining the candidate primers;

[Equation 5]

(W _l , W _g , W _t , W _h , W _d and W _e in Equation 5 are weights for primer length, %gc, Tm, hairpin formation, dimer formation, and primer entropy, respectively. In this case, each weight is W _l , W _g , W _t , W _h , W _d and W _e can be defined by the user and have a value of 0 or more, and the initial value is set to 1.

l _f and l _r are the lengths of the forward and reverse primers, respectively, and l _o is the optimal primer length by user definition.

gc _f and gc _r are %gc of the forward and reverse primers, and gc _o is the optimal %gc by user definition.

h _f , h _r is the hairpin maximum score of the forward and reverse primers,

d _f , d _r is the maximum score of the dimer (dimer) of the forward and reverse primers, and

φ _f , φ _r are primer entropy for forward and reverse primers calculated in Equation 4 above)

Even if the entropy is low, properties suitable as a primer must be satisfied. In the present invention, for this purpose, it is possible to select a primer set based on a reference value set by the experimenter by reflecting any one or more of the length of the primer, the GC fraction, the hairpin score, the dimer score, and the Tm value.

Length score = | Optimal length-primer length |

GC fraction = | Optimal GC%-primer GC% |

hairpin score = Max value (3' hairpin formation), (A/T =2, G/C=4)

dimer score = Max value (dimer formation), (A/T =2, G/C=4)

Tm score = | Optimal Tm-primer Tm |

The use of a primer that is too long may form a primer dimer or have more sequences that have room for binding to the wrong part of the template, resulting in non-specific primer-template binding, so it is preferable to select an appropriate length. . In the present invention, the primer length may be adopted in the range of 10-50 bp, preferably in the range of 10-40 bp, and most preferably in the range of 10-25 bp.

In order to have sufficient bonding strength at room temperature, it is desirable to increase the GC ratio as much as possible. Usually, the higher the GC ratio, the stronger the bonding force.

The present invention also provides a composition for amplifying a target DNA sequence comprising an oligonucleotide for amplifying a target DNA sequence according to the present invention.

The composition for amplification of a target DNA sequence according to the present invention is characterized in that it further comprises a CAS 9 enzyme that reacts with an oligonucleotide for amplification of the target DNA sequence.

In the composition for amplifying a target DNA sequence according to the present invention, the Cas 9 enzyme reacted with the oligonucleotide for DNA amplification (Catalytically Cas 9) is characterized in that the endonuclease function is lost.

In the composition for amplification of the target DNA sequence according to the present invention, the Cas 9 enzyme reacted with the oligonucleotide for DNA amplification (Catalytically Cas 9) is Natronobacteria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus , Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, and Campylobacter.

The present invention also provides a method for amplifying a target DNA sequence using an oligonucleotide for amplifying a target DNA sequence according to the present invention.

The amplification method of the target DNA sequence according to the present invention may be PCR, LAMP, or NGS.

The oligonucleotide for amplification of the target sequence according to the present invention and the method of amplifying the target sequence using the same include CRISPER-CAS9 or a gene editing system corresponding thereto, detecting a position capable of binding in the target sequence, and CAS 9 enzyme It exhibits the effect of binding and amplifying the target sequence at room temperature without special equipment through reaction with

1 shows a conventional CRISPER-CAS9 system.
2 shows the process of amplifying a target sequence according to the present invention.

A method for amplifying a target DNA sequence using an oligonucleotide for amplifying a target DNA sequence that hybridizes to a target DNA sequence and reacts with a Cas 9 enzyme (Catalytically Cas 9), wherein the oligonucleotide comprises:
A first step of performing multiple sequence alignment of a plurality of gene data;
A second step of calculating a first column entropy for each column of gene data aligned with multiple sequences in the first step;
A third step of generating forward and reverse candidate primers while changing the starting position of the primer and the length of the primer for the entire sequence from the first nucleotide sequence to the last nucleotide sequence of the multi-sequence aligned gene sequence in the second step;
For the sequence in the forward and reverse candidate primers generated in the third step, a position-specific gradient penalty value reflecting the position information in the candidate primer in the column entropy value of the second step Applying a fourth step of calculating a second column entropy;
A fifth step of calculating primer entropy for the forward and reverse candidate primers by summing the entropy of the second column at all sequence positions in the forward and reverse candidate primers;
Forward and reverse primers reflecting any one or more of the primer length, GC fraction (%gc), Tm temperature, hairpin or dimer score based on the primer entropy value of the fifth step. A sixth step of calculating a primer set matching score of; And
It characterized in that it is designed by a method comprising; a seventh step of selecting forward and reverse primers based on the primer set matching score.

Claims (12)

A method for amplifying a target DNA sequence using an oligonucleotide for amplifying a target DNA sequence that hybridizes to a target DNA sequence and reacts with a Cas 9 enzyme (Catalytically Cas 9), wherein the oligonucleotide comprises:
A first step of performing multiple sequence alignment of a plurality of gene data;
A second step of calculating a first column entropy for each column of gene data aligned with multiple sequences in the first step;
A third step of generating forward and reverse candidate primers while changing the starting position of the primer and the length of the primer for the entire sequence from the first nucleotide sequence to the last nucleotide sequence of the multi-sequence aligned gene sequence in the second step;
For the sequence in the forward and reverse candidate primers generated in the third step, a position-specific gradient penalty value reflecting the position information in the candidate primer in the column entropy value of the second step Applying a fourth step of calculating a second column entropy;
A fifth step of calculating primer entropy for the forward and reverse candidate primers by summing the entropy of the second column at all sequence positions in the forward and reverse candidate primers;
Forward and reverse primers reflecting any one or more of the primer length, GC fraction (%gc), Tm temperature, hairpin or dimer score based on the primer entropy value of the fifth step. A sixth step of calculating a primer set matching score of; And
The method characterized in that it is designed by a method comprising; a seventh step of selecting forward and reverse primers based on the primer set matching score.
The method of claim 1,
The DNA amplification oligonucleotide is 5'phosphorylated
Way.
The method of claim 1,
The DNA amplification oligonucleotide reacts with the Cas 9 enzyme (Catalytically Cas 9) is the endonuclease function is lost
Way
The method of claim 1,
The Cas 9 enzyme reacted with the DNA amplification oligonucleotide (Catalytically Cas 9)
Selected from the group consisting of Natronobacteria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburybacteria, Parvibaculum, and Niisseria, Roseburybacteria, Parvibaculum Is derived from the microorganism
Way.
The method of claim 1,
The oligonucleotide for amplifying the target DNA sequence is a DNA sequence
Way.
The method of claim 1,
In the second step, the first column entropy value is calculated according to Equation 1 below.
[Equation 1]

(In Equation 1, Pa is the gene mutation fraction at the corresponding position, K is the column height as the number of species subjected to multiple sequence processing, and i represents a specific position of the gene in the multi-sequence aligned gene data)
The method of claim 1,
The gradient penalty value η(i) in the fourth step is according to Equation 2 below.
[Equation 2]

(In Equation 2, λ is the weight of the gradient penalty, lp is the primer length, Di is the distance from the 5'end of the primer to the specific sequence position i, and τ is the inheritance at the specific sequence position i of the primer. It is the number of enemy mutation types.)
The method according to claim 6 or 7,
In the fourth step, the second column entropy value for each row in the primer is calculated according to Equation 3 below.
[Equation 3]

(In Equation 3, η(i) is a gradient penalty at a specific position i according to Equation 2, and entropy(i) is a column entropy at a specific position according to Equation 1.)
The method of claim 8,
In the fifth step, the primer entropy value is calculated according to Equation 4 below.
[Equation 4]

(In Equation 4, K is the primer length, and Q(i) is the second column entropy at a specific position i according to Equation 3)
The method of claim 9,
In the sixth step, the primer setting matching score is calculated according to Equation 5 below.
[Equation 5]

(W _l , W _g , W _t , W _h , W _d and W _e in Equation 5 are weights for primer length, %gc, Tm, hairpin formation, dimer formation, and primer entropy, respectively. In this case, each weight is W _l , W _g , W _t , W _h , W _d and W _e can be defined by the user and have a value of 0 or more, and the initial value is set to 1.
l _f and l _r are the lengths of the forward and reverse primers, respectively, and l _o is the optimal primer length by user definition.
gc _f and gc _r are %gc of the forward and reverse primers, and gc _o is the optimal %gc by user definition.
h _f , h _r is the hairpin maximum score of the forward and reverse primers,
d _f , d _r is the maximum score of the dimer (dimer) of the forward and reverse primers, and
φ _f and φ _r are the primer entropy for the forward and reverse primers calculated in Equation 4 above)
A method of amplifying a target DNA sequence using an oligonucleotide for amplifying a target DNA sequence according to claim 1
The method of claim 11,
The method of amplifying the target DNA sequence is PCR, LAMP, or NGS.
Amplification method of target DNA sequence

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