CN116064757A

CN116064757A - Probe design method for improving specificity of TaqMan probe

Info

Publication number: CN116064757A
Application number: CN202211185148.8A
Authority: CN
Inventors: 姜正文; 张德康; 余锋
Original assignee: GENESKY DIAGNOSTICS (SUZHOU) Inc; Tianhao Gene Technology Suzhou Co ltd
Current assignee: GENESKY DIAGNOSTICS (SUZHOU) Inc; Tianhao Gene Technology Suzhou Co ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-05-05

Abstract

The invention provides a design method for improving the specificity of a TaqMan probe. Specifically, mismatched bases are introduced into positions adjacent to nucleotide polymorphism sites of the TaqMan probe, so that the TaqMan probe with higher specificity is designed, and the problem of cross reaction in the SNP typing detection process can be reduced. Has application prospect in SNP typing detection.

Description

Probe design method for improving specificity of TaqMan probe

Technical Field

The present invention relates to the field of gene polymorphism detection. In particular, the invention relates to a probe design method for improving the specificity of TaqMan probes.

Background

Nucleotide polymorphism (Nucleotide Polymorphism) refers to a polymorphism in a DNA sequence at the genomic level caused by nucleotide variation. The presence of nucleotide polymorphism sites often causes changes in genes, particularly single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNPs) that can lead to the development of certain diseases, such as various genetic diseases, malignancies, etc. With the rapid development of molecular biology in recent years, more and more disease-related genes have been discovered. Therefore, detection of mutant genes has a significant meaning for diagnosis and risk prediction of diseases, and more mutant gene detection techniques have been developed. The current methods for detecting gene mutation mainly comprise a DNA sequencing method, a mutation amplification blocking system (Amplification Refractory Mutation System, ARMS), a real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) method and the like.

DNA sequencing methods commonly used today are first generation sequencing and second generation sequencing. The Sanger sequencing method is the most reliable method for detecting SNP loci of genes, and is a gold standard in medicine, but the method is complex in operation and high in cost, so that the method is not suitable for detecting a large number of clinical samples. The second generation sequencing has high flux, large gene coverage and large sequencing depth, but the detection cost of the known mutation sites is relatively high, and special reagents, instruments and personnel are required for carrying out experiments and analyzing data, so that the detection is complex.

In the last 90 th century, qPCR technology was introduced by ABI, U.S.A., a method of detecting the total amount of product after each cycle with fluorescent chemicals during PCR amplification. The qPCR method is used for detecting nucleotide polymorphism, and a pair of TaqMan probes with different fluorescent labels at the 5' end are added to identify different alleles during PCR reaction, and different genotypes are distinguished through the combination of fluorescent signals. The method is not only efficient and quick for detecting the known specific mutation sites, but also can distinguish different genotypes, and is an ideal method. However, in the actual operation process, the mutation type and the wild type template have only one base difference, so that the cross reaction is easy to generate, and the detection result is interfered to a certain extent.

So far, studies have shown that introduction of mismatched bases on primers can often improve the specificity of PCR reactions, but for TaqMan probes, there is no precedent for introduction of mismatched bases. Therefore, there is an urgent need to develop a method for improving the specificity of such TaqMan probes.

Disclosure of Invention

The invention aims to provide a probe design method for improving the specificity of a TaqMan probe.

In a first aspect of the present invention, there is provided a TaqMan probe for detecting a nucleotide polymorphism, which specifically binds to a target sequence in which a polymorphic site is located, and which has mismatched bases 1 to 5 bases from the base to which the polymorphic site is bound.

In another preferred embodiment, the mismatched base forms a mismatch with the base at the position corresponding to the target sequence selected from the group consisting of: A/G, A/C, A/A, T/G, T/C, T/T, G/G, C/C, or a combination thereof.

In another preferred embodiment, there is at least 1 (preferably 1-5, more preferably 1-3, more preferably 1-2) base mismatch between the probe and the target sequence.

In another preferred embodiment, the mismatched base is located 1-5 bases, preferably 1-3 bases, more preferably 1-2 bases, and even more preferably 1 base from the base of the binding polymorphic site.

In another preferred embodiment, the probe is used to detect Single Nucleotide Polymorphisms (SNPs).

In a second aspect of the present invention, there is provided a TaqMan probe combination for detecting nucleotide polymorphisms, the probe combination comprising probe A and probe B, wherein probe A specifically binds to a wild-type genotype target sequence, probe B specifically binds to a mutant genotype target sequence, and

the probe a and/or probe B is a probe according to the first aspect of the invention.

In another preferred embodiment, the wild-type and mutant-type target sequences have at least 1, preferably 1, different base.

In another preferred embodiment, there are at least 2 (preferably 2-5, more preferably 2-3) base mismatches between the probe A and the mutant genotype target sequence, and/or at least 2 (preferably 2-5, more preferably 2-3) base mismatches between the probe B and the wild-type genotype target sequence.

In another preferred embodiment, the probe binds to a template strand, or coding strand, of the target sequence.

In another preferred embodiment, the 5' ends of the probe A and the probe B are respectively connected with different fluorophores.

In another preferred embodiment, the probe combination is used to detect Single Nucleotide Polymorphisms (SNPs).

In a third aspect of the invention there is provided the use of a probe according to the first aspect of the invention, or a combination of probes according to the second aspect of the invention, for the preparation of a reagent or kit for detecting nucleotide polymorphisms.

In another preferred embodiment, the reagent or kit further comprises a primer.

In another preferred embodiment, the reagent or kit is used for qPCR detection.

In another preferred embodiment, the reagent or kit is used to detect Single Nucleotide Polymorphisms (SNPs).

In a fourth aspect of the present invention, there is provided a method of preparing a probe according to the first aspect of the present invention, or a probe combination according to the second aspect of the present invention, comprising the steps of:

(i) Designing a probe A and a probe B aiming at the detected wild type site and the detected mutant site respectively;

(ii) Mismatched bases are introduced on probe A and/or probe B1-5 bases from the base of the binding polymorphic site.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

Fig. 1 shows a technical principle view of the present invention.

FIG. 2 shows a schematic diagram of amplification results without introducing a mutant base to the G allele TaqMan probe in the examples of the present invention.

FIG. 3 shows a schematic diagram of amplification results of introduction of mismatched bases to the 1 st position on the left side of SNP site of G allele TaqMan probe in the embodiment of the invention.

FIG. 4 shows a schematic diagram of amplification results of introducing mismatched bases to the 2 nd left of the SNP site of the G allele TaqMan probe in the embodiment of the invention.

FIG. 5 shows a schematic diagram of amplification results of introduction of mismatched bases to the 1 st right side of the SNP site of the G allele TaqMan probe in the embodiment of the invention.

Detailed Description

Through extensive and intensive research, the inventor develops a design method for improving the specificity of the TaqMan probe for the first time, and a TaqMan probe with higher specificity is designed by introducing mismatched bases at the position adjacent to the SNP locus of the TaqMan probe, so that the problem of cross reaction in the SNP typing detection process can be reduced. On this basis, the present invention has been completed.

Terminology

As used herein, the term "nucleotide polymorphism (Nucleotide Polymorphism)" refers to a DNA sequence polymorphism caused by nucleotide variation at the genomic level. The nucleotide polymorphism referred to in the present invention includes sequence polymorphism caused by base substitution, insertion or deletion of one or more bases. The present invention provides a method for detecting nucleotide polymorphisms by qPCR using the probe or probe combination of the present invention. The probe or probe combination of the present invention can be used to detect any nucleotide polymorphism. In a preferred embodiment, the probe or combination of probes of the invention is used to detect single nucleotide polymorphisms. In another embodiment, the probe or probe combination of the present invention can be used to detect a sequence polymorphism generated by a base substitution, insertion or deletion of one or more bases.

As used herein, the term "single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP)" refers to a DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. SNP is generally composed of only two bases, so it is a binary marker, i.e., a binary gene (binary). Because of the binary nature of SNPs, not just, SNPs often require only +/-analysis in genomic screening, which is also advantageous for genotyping. Typically, there is often a predominant allele in the natural population, known as the wild-type gene. In contrast, mutant genes.

As used herein, the terms "cross-reactive", "non-specific reactive" and "non-specific binding" are used interchangeably to refer to the non-specific reaction of a probe, i.e., the non-specific binding of a probe to other sequences having high homology to its target sequence. Typically, the cross-reaction to be overcome by the present invention refers to the non-specific binding that occurs between the templates of probe A and probe B, or between probe B and probe A during PCR.

As used herein, the term "mismatched base" refers to the pairing of adenine (a) with cytosine (C) or guanine (G), or thymine (T) with cytosine (C) or guanine (G), or a pair of adenine (a), a pair of thymine (T), a pair of cytosine (C), or a pair of guanine (G) in a DNA duplex.

Probe with a probe tip

As used herein, the term "probe" refers to a small single-stranded DNA or RNA fragment used to detect a nucleic acid sequence complementary thereto. In the probe of the present invention, the nucleotide polymorphism site is preferably located at the middle position of the probe.

The present invention aims to overcome non-specific binding that occurs between probes and non-target templates. This binding is caused by base mismatches. The probability of occurrence of mismatch varies among different bases, and therefore, mismatched bases are preferably introduced into a probe that is liable to be mismatched. For example, the present invention has found that G-T base pairs are more prone to mismatches. Thus, in a preferred embodiment of the invention, mismatched bases are introduced for probes that form G-T mismatches at the SNP site. Of course, it will be appreciated by those skilled in the art that with the probe combinations of the present invention, mismatched bases can be introduced into both probes, thereby increasing their specificity.

Typically, FIG. 2 shows the amplification results without introducing a mutant base to the G allele TaqMan probe. Among them, it was observed that a probe for detecting a G allele (G probe) was more likely to form a G-T base mismatch at the mutation site, whereas a probe for detecting an A allele (A probe) was less likely to generate an A-C base mismatch. In this case, in one embodiment, the introduction of mismatched bases only in the G probe increases its specificity, while the A probe does not introduce mismatched bases. In another embodiment, mismatches may be introduced in both the G and A probes to increase their specificity.

Detection method

qPCR technology is a method of detecting the total amount of product after each cycle with fluorescent chemicals during PCR amplification. The qPCR method is used for typing nucleotide polymorphic sites, and during PCR reaction, a pair of TaqMan probes with different fluorescent labels at the 5' end are added to identify different alleles, and different genotypes are distinguished through the combination of fluorescent signals.

The detection method of the invention utilizes the specially designed probe, and if the probe is combined with the non-specific template in the annealing process of the probe and the template, the combination efficiency is greatly reduced due to at least 2 mismatched bases, so that the combination specificity and detection efficiency of the TaqMan probe are improved.

The specific TaqMan probe design scheme and specific detection steps are as follows:

(1) The nucleotide polymorphism site is positioned in the middle of the probes, the 5 'end of each probe is provided with different fluorescent marking groups, the 3' end is provided with a fluorescent quenching group, and mismatched bases are introduced between 1-5 bases on the left side or/and the right side of the nucleotide polymorphism site of the probe;

(2) Designing a pair of common PCR primers according to the region where the detected nucleotide polymorphism site is located;

(3) Mixing a common PCR primer group with TaqMan probes of two different alleles designed by the TaqMan design method according to a certain proportion;

(4) And (3) configuring a reaction system, amplifying in a fluorescent quantitative PCR instrument and collecting fluorescent signals in real time.

The main advantages of the invention include:

(1) The detection speed is high: the whole PCR reaction is carried out on a fluorescent quantitative PCR instrument, and the result can be interpreted through fluorescent signal data after the reaction is finished;

(2) Genotypes can be distinguished: after the PCR reaction is finished, different gene types of the sample can be distinguished through different fluorescent combinations, and wild type, heterozygous and homozygous mutations can be distinguished;

(3) The specificity is high: the specificity of the probe is improved by introducing mismatched bases at the adjacent positions of the nucleic acid polymorphic sites of the TaqMan probe, and the binding efficiency is greatly reduced due to the existence of at least 2 mismatched bases when the probe is combined with a non-specific template in the annealing process of the probe and the template, so that the binding specificity of the TaqMan probe is improved.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1

In the invention, the rs2274223 locus is taken as an example, and because the locus is of an A/G mutation type, in the actual detection process, a probe (gray fluorescent marker) for detecting the G allele can generate nonspecific binding (as shown in figure 2), so that the embodiment improves the specificity of the probe by introducing mismatched bases on the probe.

The invention is used for detecting 3 DNA samples with the genotypes of the rs2274223 locus of AA, AG and GG respectively. Mainly comprises the following steps:

(1) TaqMan probe designUnderline lineThe marker is a SNP site,double underlineTag as the mismatched base introduced):

designing a common TaqMan probe aiming at the detected allele A, wherein the 5 'end of the probe is provided with a fluorescent group FAM, the 3' end of the probe is provided with a fluorescence quenching group BHQ1, and the sequence is rs2274223-TaqMan-probe1: FAM-CAGTTTCTGTTCCACGTTCACTTCG-BHQ1(SEQ ID NO.1)。

Designing a TaqMan probe with mismatched bases introduced at the 1 st position on the left side aiming at detecting an allele G, wherein the 5 'end is provided with a fluorescent group JOE, the 3' end is provided with a fluorescence quenching group BHQ1, and the sequence is rs2274223-TaqMan-probe 2: JOE-AGCAGTTTCTGTTC

GCGTTCACTTCGAAG-BHQ1(SEQ ID NO.2)。

In order to increase contrast, the invention designs a probe rs2274223-TaqMan-probe3-1 without introducing mismatched bases for detecting the allele G: JOE-AGCAGTTTCTGTTCCGCGTTCACTTCGAAG-BHQ1 (SEQ ID NO. 3), and a probe rs2274223-TaqMan-probe3-2 with mismatched base introduced at the 2 nd position on the left: JOE-AGCAGTTTCTGTT

CGCGTTCACTTCGAAG-BHQ1 (SEQ ID NO. 4), and a probe rs2274223-TaqMan-probe3-3 with mismatched base introduced at position 1 on the right: JOE-AGCAGTTTCTGTTCCG->

GTTCACTTCGAAG-BHQ1(SEQ ID NO.5)。

(2) Primer design:

a pair of PCR primers is designed aiming at the locus rs 2274223:

the upstream primer rs2274223-5F:5'-TCGAAACACCCTGAACCCCATGT-3' (SEQ ID NO. 6);

the downstream primer rs2274223-3R:5'-GTTTTCCACAACTGCAAAACGAAGAAA-3' (SEQ ID NO. 7).

(3) Primer set configuration:

configuration concentration of primer set Pmix: 2. Mu.M of upstream PCR primer, 2. Mu.M of downstream PCR primer, 1. Mu.M of detection allele A TaqMan probe and 1. Mu.M of detection allele G TaqMan probe.

(4) The reaction system:

the PCR reaction system uses 30 mu L of system reaction components, and the components are respectively as follows: 10 XTakara Buffer 3. Mu. L, dNTP (2.5 mM) 3.6. Mu. L, mgCl2 (25 mM), pmix 1.5. Mu.L, takara Taq (5U/. Mu.L) 0.18. Mu.L, sample DNA 1. Mu.L (20 ng) and ddH ₂ O 19.52μL。

PCR reaction procedure: 95℃for 10min,40× (15 s at 95℃and 40s at 64℃for collecting fluorescence signals).

Results: application of

Data analysis was performed by 96S fully automated medical PCR analysis system software, with results shown in FIGS. 2-5 (FAM in black and JOE in gray).

In an ideal state, the AA genotype has only FAM fluorescence peak (black) or FAM fluorescence Ct value far less than JOE fluorescence (gray), the AG genotype has FAM and JOE fluorescence peak at the same time and Ct value near, and the GG genotype has only JOE fluorescence peak or JOE fluorescence Ct value far less than FAM.

As shown in fig. 2, in the AA genotype result, the G probe cross-reacts with the template of allele a, thereby generating a fluorescent signal, causing a large interference to detection, and having Ct values of 31.43 and 31.38 for FAM and JOE, respectively, without significant difference; in the AG genotype detection results, the difference in Ct values between FAM and JOE was also large, 33.10 and 30.88, respectively. After introduction of mismatched bases, this non-specific binding efficiency is greatly reduced (shown in FIGS. 3, 4 and 5). In this example, the effect of introducing mismatched base at the 1 st position and mismatched base at the 1 st position on the right side of the SNP locus of the probe is best (as shown in FIG. 3 and FIG. 5), and the difference between the fluorescence signal Ct values of FAM and JOE in the AA genotype is the largest, which are 29.36, 31.64, 30.52 and 32.88 respectively; the two Ct values in the AG genotype were also closest, 32.07, 31.83 and 33.06, 32.90, respectively.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A TaqMan probe for detecting a nucleotide polymorphism, characterized in that the probe specifically binds to a target sequence in which a polymorphic site is located, and that the probe has mismatched bases 1 to 5 bases from the base to which the polymorphic site is bound.

2. The probe of claim 1, wherein the mismatched base forms a mismatch with a base at a position corresponding to the target sequence selected from the group consisting of: A/G, A/C, A/A, T/G, T/C, T/T, G/G, C/C, or a combination thereof.

3. Probe according to claim 1, characterized in that there is at least 1 (preferably 1-5, more preferably 1-3, more preferably 1-2) base mismatch between the probe and the target sequence.

4. The probe of claim 1, wherein the mismatched base is located 1-5 bases, preferably 1-3 bases, more preferably 1-2 bases, and even more preferably 1 base from the base of the binding polymorphic site.

5. A TaqMan probe combination for detecting nucleotide polymorphism is characterized by comprising a probe A and a probe B, wherein the probe A specifically binds to a wild type genotype target sequence, the probe B specifically binds to a mutant genotype target sequence, and

the probe a and/or probe B is the probe according to any one of claims 1 to 4.

6. The probe combination according to claim 5, wherein the wild-type and mutant genotype sequences have at least 1 different base, preferably 1 different base.

7. The probe combination of claim 5, wherein at least 2 base mismatches exist between the probe a and the mutant genotype target sequence and/or at least 2 base mismatches exist between the probe B and the wild-type genotype target sequence.

8. The probe assembly of claim 5, wherein the 5' ends of probe A and probe B are each attached to a different fluorophore.

9. Use of a probe according to claim 1, or a combination of probes according to claim 5, for the preparation of a reagent or kit for detecting nucleotide polymorphisms.

10. The use according to claim 9, wherein the reagent or kit is for detecting Single Nucleotide Polymorphisms (SNPs).

11. A method of preparing a probe according to claim 1 or a probe combination according to claim 5, comprising the steps of:

(i) Respectively designing probes aiming at the detected wild type locus and the detected mutant locus;

(ii) Mismatched bases are introduced on the probe 1-5 bases from the base that binds the polymorphic site.