CN111334562A - Nucleic acid amplification method and kit containing modified primer - Google Patents
Nucleic acid amplification method and kit containing modified primerDownload PDFInfo
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Abstract
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nucleic acid amplification method containing a modified primer and a kit.
Background
Nucleic acid amplification techniques are commonly used for nucleic acid-based detection, such as analyte detection, sensing, forensic and diagnostic applications, genome sequencing, whole genome amplification, and the like.
Currently, a variety of techniques are available for amplifying nucleic acids, including Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), autonomous sequence replication system (3SR), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), and Rolling Circle Amplification (RCA).
In the case of PCR technology, the basic principle of nucleic acid amplification is an enzymatic synthesis reaction that relies on a thermostable DNA polymerase in the presence of template DNA, primers and 4 deoxymononucleotides. In PCR, a DNA to be amplified is used as a template, two oligonucleotides which are complementary to the ends of a positive strand and a negative strand of the template are used as primers, and a new template DNA is synthesized by template DNA denaturation, template primer renaturation combination and primer chain extension reaction under the action of DNA polymerase. Thus, the amount of the DNA of interest will accumulate in an exponentially increasing fashion, and the segment of the template between the two primers will be amplified continuously. The amplification product can be detected by gel electrophoresis, Southern hybridization or DNA sequence analysis.
The current common nucleic acid amplification technology can simply, conveniently and quickly obtain a large amount of specific nucleic acid from a trace amount of biological materials in an in-vitro amplification mode, and can be used for detecting trace amount of samples in animal quarantine. However, there are significant disadvantages in the nucleic acid amplification process, such as susceptibility to high background signal during nucleic acid amplification, non-specific amplification bands, sheet-like bands or smears, which may be caused by: amplification of contaminating nucleic acids in the sample, primer dimer formation, or incomplete complementary primer to target sequence, independent primer-primer interactions, and the like. The occurrence of these problems hampers the analytical detection of nucleic acids and can lead to incorrect diagnostic results, such as the diagnosis of false positive results.
Disclosure of Invention
To solve the above problems, the present invention provides a nucleic acid amplification method and kit comprising modified primers having high affinity for natural oligonucleotides but little affinity for other oligonucleotides, even non-natural oligonucleotides of complementary sequence, by adding primers containing modified bases to base-pair-bind to natural DNA. The addition of a primer containing a modified base makes it possible to suppress the generation of non-specific amplification bands such as undesired primer dimers and chimeric products, and to amplify a desired target nucleic acid, for example, a "DNA template" or a "nucleic acid template" more efficiently.
In a first aspect, the present invention provides a method for amplifying a nucleic acid containing a modified primer, the method for amplifying a nucleic acid containing a modified primer, comprising:
(i) providing a primer comprising a modified base, a dNTP, a nucleic acid template comprising a target sequence at least partially complementary to said primer, an enzyme for processing the template, and a buffer solution; wherein the primers comprise a forward primer and a reverse primer;
(ii) providing conditions suitable for amplification of nucleic acid by a primer, a nucleic acid template, an enzyme, and contacting the nucleic acid template with the primer;
(iii) and carrying out amplification reaction, carrying out pairing combination on the primer and a complementary sequence on the nucleic acid template, and extending and amplifying the nucleic acid template.
Preferably, the primer is about 8 to 25 nucleotides in length.
Preferably, the modification position of the modified base is selected from C-5 of pyrimidine and C-2 of purine, wherein the substituent of the modification position comprises methyl, propynyl and amino.
Preferably, the modified base comprises a thymine replaced with 5-propynyl-2' -deoxyuridine.
Preferably, the primer comprises at least one modified base, wherein the modified base comprises 5-methyl-2 ' -deoxycytosine, 5-propynyl-2 ' -deoxyuridine, 2-amino-deoxyadenosine, 2-amino-7-deaza-2 ' -deoxyadenosine; wherein the incorporation of the substituent in the 5-methyl-2 '-deoxycytidine, 5-propynyl-2' -deoxyuridine improves the stability of the double strand binding to the template and the Tm value of the primer; wherein incorporation of the substituent in the 2-amino-deoxyadenosine, 2-amino-7-deaza-2' -deoxyadenosine prevents primer dimer formation.
Preferably, the primer comprises a Dickerson-Drew dodecamer with the sequence of [ CGCGAATTCGCG ]2。
Preferably, the dntps include, but are not limited to: dATP, dCTP, dGTP and dUTP.
Preferably, the strand of the nucleic acid template has a sequence comprising a forward primer and a reverse primer binding site.
Preferably, the amplification conditions comprise isothermal amplification.
Preferably, the amplification reaction is carried out in a 100ul system.
Preferably, the procedure of the amplification reaction is:
the above procedure is specifically: pre-denaturation at 50 ℃ for 5min, denaturation at 98 ℃ for 20 sec, annealing at 62 ℃ for 20s, extension at 72 ℃ for 45s, and cycling for 41 times, and finally extension at 72 ℃ for at least 10 min.
Preferably, the amplification reaction comprises amplification in a nested PCR format, a digital PCR format, or a multiplex PCR format.
Preferably, the forward and reverse primers in the amplification reaction are more complementary to the primer binding site on the template than to the primer binding site.
Preferably, the amplification reaction comprises at least 2 pairs of forward and reverse primers, wherein the primers are useful for multiplex amplification and different primers are attached to corresponding target sequences having different melting temperatures for hybridizing to multiple primer binding sites at different positions in the target nucleic acid template and forming multiple extension fragments.
Further preferably, the plurality of primers are contacted with the nucleic acid template at different concentrations.
In a second aspect, the present invention provides a kit comprising: a primer comprising a modified base as described in the first aspect, a dNTP, a nucleic acid template comprising a target sequence at least partially complementary to said primer, an enzyme and buffer solution for processing the template, and a fluorophore for detection.
Preferably, the kit is connected with the oligonucleotide marked by the fluorescent signal group and the quenching fluorescent group through at least one pair of primers; wherein the two ends of the oligonucleotide are labeled by a fluorescent group and a quenching fluorescent group, and the fluorescent group is separated from the quenching fluorescent group during amplification to form a fluorescent molecule, which indicates that an amplification product of the nucleic acid template exists.
Preferably, the kit comprises a dodecamer, wherein the dodecamer comprises 2-amino-deoxyadenosine, 5-methyl-2 ' -deoxycytidine, 5-propynyl-2 ' -deoxyuridine, and 2-amino-7-deaza-2 ' -deoxyadenosine, and is used for a nucleic acid amplification reaction.
In a third aspect, the present invention provides a nucleic acid amplification method comprising a modified primer according to the first aspect and a kit according to the second aspect, for use in detecting nucleotide insertions, deletions, rearrangements, transitions, translations, transversions, polymorphisms and substitutions.
The invention provides a nucleic acid amplification method containing a modified primer and a kit, which have the beneficial effects that: by adding the primer containing the modified base to be paired with the natural DNA base, the affinity and stability of the base pairing of the modified base and the natural DNA are improved, and the affinity of the modified base and the unnatural DNA base is inhibited. The nucleic acid amplification method and the kit containing the modified primer provided by the invention effectively reduce the generation of non-specific amplification bands such as primer dimers, more effectively amplify the required target nucleic acid and enable the detection result to be more accurate.
Drawings
FIG. 1: are standard base pairs describing the modified bases 5-methyl-2 '-deoxycytidine, 5-propynyl-2' -deoxycytidine, unmodified deoxycytidine of the invention.
FIG. 2: the unmodified standard base T, the standard base T-A base pair and the modified base 5-propynyl-2' -deoxyuridine of the invention are described.
FIG. 3: describes the base pairing combination of the modified base 2-amino-7-deaza-2' -deoxyadenosine (2-amino-7-deaza-dA) of the invention instead of the base dA and the standard base T, the standard base T-A base pair.
FIG. 4: gel electrophoresis images of PCR amplification reactions.
Detailed Description
The present invention is further described in conjunction with the following detailed description, which is given by way of illustration only, and not for the purpose of limiting the scope of the invention as many variations thereof are possible without departing from the spirit and scope thereof. It will be understood that no inventive modifications will occur to those skilled in the art upon reading the specification in light of conventional requirements, but which are protected by the patent laws within the scope of the appended claims.
In a first aspect, a method of nucleic acid amplification comprising a modified primer is described, as shown in FIG. 1, wherein the base dC is replaced with a modified base 5-methyl-2 ' -deoxycytidine, 5-propynyl-2 ' -deoxycytidine, and the introduction of a group at the C-5 position enhances the hydrophobic nature of the base to facilitate the elimination of water molecules from the duplex by base complementary pairing, and the 5-propynyl-2 ' -deoxycytidine-containing oligodeoxynucleotide primer significantly enhances the duplex formation of single stranded DNA and RNA.
The modified base 5-propynyl-2' -deoxyuridine is used for replacing the base dT, and the propynyl with hydrophobicity is introduced by modifying the C-5 group, so that the removal of water molecules from double-stranded molecules is facilitated, the double helix formation of single-stranded DNA and RNA is enhanced, and the increase of hybridization efficiency is facilitated.
Example 1: description of the materials
The amplification reactions shown in Table 1 use primers from the region of the 16S rRNA gene of M.tuberculosis, the primers being a forward primer and a reverse primer, the primers being modified or unmodified; specifically, the primers of the present invention are shown in Table 1 below.
TABLE 1 PCR primer sequences
Example 2: nucleic acid amplification assay
The invention provides a nucleic acid amplification method containing a modified primer, which comprises the following steps:
amplification was performed in a 100ul system using DNA from Mycobacterium tuberculosis as a template DNA, using the primers in Table 1, including the forward primer and the reverse primer, in reactions consisting of 50mM Tris-HCl (pH8.9), 50mM KCl, 100. mu. dATP, 100. mu.M dCTP, 100. mu.M dGTP, 100. mu.M dUTP, 250nM of each primer in Table 1, 5 units of polymerase, 10% glycerol;
amplification and analysis were done using a LightCycler 480 instrument, and amplification was performed using the PCR instrument program set up with the following conditions:
PCR reaction set-up procedure:
the above procedure is specifically: pre-denaturation at 50 ℃ for 5min, denaturation at 98 ℃ for 20 sec, annealing at 62 ℃ for 20s, extension at 72 ℃ for 45s, and cycling for 41 times, and finally extension at 72 ℃ for at least 10 min.
The temperature of 72 ℃ is kept constant for more than 10min in an Applied Biosystems DNA9600 type thermal cycler, after the reaction is finished, a small amount of samples are taken to analyze an amplification product through 0.5% agarose gel electrophoresis, and then ethidium bromide staining is carried out. The images were visualized by Typhoon imager scanning, as shown in figure 4.
As shown in FIG. 4, the numbers 1-8 correspond to the primer numbers of Table 1, and the target fragments are shown in lanes 1-8; m is Marker.
Wherein the incorporation of 2-amino-deoxyadenosine, 2-amino-7-deaza-2' -deoxyadenosine increases the electrostatic effect of the primers, preventing primer dimer formation; among them, the lanes using 2-amino-7-deaza-2' -deoxyadenosine as a modified base show significantly fewer non-specific amplification bands, are bright in color, and more effectively inhibit the formation of non-specific amplification bands such as primer dimers.
Example 3: measurement of Tm value
Base modification is carried out on an unmodified primer by using a base 5-methyl-2 '-deoxycytidine, a base 5-propynyl-2' -deoxycytidine to replace a base dC and a base 5-propynyl-2 '-deoxyuridine to replace a base dT, so as to obtain a primer containing modified bases 5-methyl-2' -deoxycytidine, 5-propynyl-2 '-deoxycytidine and 5-propynyl-2' -deoxyuridine; the primers formed are shown in Table 2 below.
Specifically, the primer is from the region of the 16S rRNA gene of Mycobacterium tuberculosis, and the primer is modified or unmodified.
TABLE 2 primers containing unmodified, C-5 modified and C-2 modified bases
| Primer sequences | Base |
| 5'-ATGTTCACTACGCGAATATGC-3' | - |
| 5'-ATGTTCACTACGCGAATXTGC-3' | 2-amino-deoxyadenosine |
| 5'-ATGTTCACTACGCGAATATGY-3' | 5-methyl-2' -deoxycytidine |
| 5'-ATGTTCACTACGYGAATATGY-3' | 5-propynyl-2' -deoxycytidine |
| 5'-ATGTZCACZACGCGAATAZGC-3' | 5-propynyl-2' -deoxyuridine |
The primer heating dissolution was carried out in a LightCycler 480 apparatus, and the complementary primer pairs shown in Table 2 were mixed at 0.25mM or 2.5mM in a buffer solution containing 10mM Tris-HCl (pH8.3), 50mM KCl, 100mM TTP,2mM MgCl 2 and 10 nMSITOVR 13 dyes;
the temperature was set to rise from 40 ℃ to 90 ℃ over 25 minutes and fluorescence was continuously monitored (excitation 465nm, emission 510 nm). Melting curves characteristic of decrease in fluorescence due to strand separation were measured, and Tm values were obtained from the first derivative of the fluorescence curves, and the Tm values for each primer are shown in Table 3.
The Tm values of the primers containing different modified bases as compared with those of the unmodified primers are shown in Table 3. As can be seen from Table 3, the incorporation of substituents in 2-amino-deoxyadenosine, 5-methyl-2 '-deoxycytidine, 5-propynyl-2' -deoxyuridine enhanced the hydrophobicity of the primer, increased the stability of the double strand binding to the template and the Tm of the primer.
TABLE 3 modified bases and corresponding Tm values
Claims (10)
1. A method for amplifying a nucleic acid containing a modified primer, comprising:
(i) providing a primer comprising a modified base, a dNTP, a nucleic acid template comprising a target sequence at least partially complementary to said primer, an enzyme for processing the template, and a buffer solution; wherein the primers comprise a forward primer and a reverse primer;
(ii) providing conditions suitable for amplification of nucleic acid by a primer, a nucleic acid template, an enzyme, and contacting the nucleic acid template with the primer;
(iii) and carrying out amplification reaction, carrying out pairing combination on the primer and a complementary sequence on the nucleic acid template, and extending and amplifying the nucleic acid template.
2. The method of claim 1, wherein the primer is about 8 to 25 nucleotides in length.
3. The method for amplifying a nucleic acid according to claim 1, wherein the modified position of the modified base is selected from the group consisting of C-5 of pyrimidine and C-2 of purine.
4. The method of claim 1, wherein said modified base comprises a thymine substituted with 5-propynyl-2' -deoxyuridine.
5. The method of claim 1, wherein the primer comprises at least one modified base, and wherein the modified base comprises 5-methyl-2 ' -deoxycytidine, 5-propynyl-2 ' -deoxyuridine, 2-amino-deoxyadenosine, or 2-amino-7-deaza-2 ' -deoxyadenosine.
6. The method of claim 1, wherein the amplification conditions comprise isothermal amplification.
7. The method of claim 1, wherein at least 2 pairs of forward and reverse primers are included in the amplification reaction, wherein the primers are used for multiplex amplification, and wherein different primers are ligated to corresponding target sequences having different melting temperatures, and are used to hybridize to multiple primer binding sites at different positions in the nucleic acid template and form multiple extension fragments.
8. A kit, comprising: the modified base-containing primer of claim 1, dntps, a nucleic acid template comprising a target sequence at least partially complementary to the primer, enzymes and buffer solutions for processing the template, and a fluorophore for detection.
9. The kit of claim 8, wherein the kit is linked to the oligonucleotide labeled with the fluorescent signal group and the quencher fluorescent group through at least one pair of primers; the fluorophore separates from the quenched fluorophore during amplification to form a fluorescent molecule, indicating the presence of the amplification product of the nucleic acid template.
10. A kit according to claim 8, wherein said kit comprises a dodecamer, wherein said dodecamer comprises 2-amino-deoxyadenosine, 5-methyl-2 ' -deoxycytosine, 5-propynyl-2 ' -deoxyuridine and 2-amino-7-deaza-2 ' -deoxyadenosine, and is used for nucleic acid amplification reaction.
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Cited By (1)
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| CN114277103A (en)* | 2022-01-21 | 2022-04-05 | 杭州飞时达生物科技有限公司 | Efficient rolling circle amplification method based on hexamer random primer |
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