PCR PRIMER LINKED TO COMPLEMENTARY NUCLEOTIDE SEQUENCE OR COMPLEMENTARY NUCLEOTIDE SEQUENCE INCLUDING MIS-MATCHED NUCLEOTIDES AND METHOD FOR AMPLIFYING NUCLEIC ACID USING THE SAME
20180002746 · 2018-01-04
Inventors
Cpc classification
C12Q1/6848
CHEMISTRY; METALLURGY
C12Q2525/161
CHEMISTRY; METALLURGY
C12Q2525/161
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a primer for PCR obtained by, directly or through inosine as a linker, linking a complementary nucleotide sequence or a complementary nucleotide sequence including a mis-matched nucleotide sequence to the 5′-terminal of a forward or reverse primer; and to a PCR method including a step of mixing a nucleic acid template in a PCR composition including the primer and then performing PCR on the mixture. The primer for PCR of the present invention includes a complementary nucleotide sequence or a mis-matched nucleotide sequence in a complementary nucleotide sequence, which is linked to the 5′-terminal thereof directly or via a linker, thereby lowering the sensitivity increase due to the increase in amplification products and reducing non-specifically occurring reactions in PCR.
Claims
1. A primer for gene amplification represented by the following Structural Formula 1, the primer comprising complementary double strands of nucleotides with a bubble structure by including a mis-matched nucleotide linked, directly or by an inosine linker, to 5′ terminal of a primer for a corresponding sequence for amplifying a specific gene sequence: ##STR00002## wherein the primer includes a complementary nucleotide sequence X.sub.a of the corresponding sequence for amplifying the specific gene sequence and a mis-matched nucleotide sequence X′.sub.a which is complementary to X.sub.a, in which X.sub.a and X′.sub.a are linked with a nucleotide sequence Y.sub.a of a universal base or a non-discriminatory base.
2. The primer of claim 1, wherein the primer has the bubble structure of the mis-matched nucleotide linked with at least one double-strand sequence.
3. The primer of claim 2, wherein the primer has the bubble structure of the mis-matched nucleotide linked with one to four double-strand sequences.
4. The primer of claim 1, wherein a double-strand of X′.sub.a and X.sub.a of the primer includes at least one bubble structure.
5. The primer of claim 4, wherein the double-strand of X′.sub.a and X.sub.a of the primer includes one to three bubble structures.
6. The primer of claim 1, wherein the number of the mis-matched nucleotide in the complementary nucleotide sequence (X′.sub.a) of the primer is at least one.
7. The primer of claim 6, wherein the number of the mis-matched nucleotide in the complementary nucleotide sequence (X′.sub.a) of the primer is 3 to 12.
8. The primer of claim 1, wherein the primer has the bubble structure including at least 0 universal base or non-discriminatory base in Y.sub.a of the primer.
9. The primer of claim 8, wherein the primer has the bubble structure including 0 to 9 universal bases or non-discriminatory bases in Y.sub.a of the primer.
10. The primer of claim 1, wherein the primer is one or more primers selected from the group consisting of the primers as set forth in SEQ ID NOS: 3 to 15, SEQ ID NOS: 18 and 19, and SEQ ID NOS: 22 to 29.
11. The primer of claim 1, wherein the primer is used in each of forward and reverse positions for gene amplification or in both.
12. A method of amplifying a specific gene sequence from RNA, wherein cDNA is synthesized with a reverse transcription polymerase for reverse transcription using the primer of claim 1, and the specific gene is amplified with the cDNA as a template using the primer of claim 1.
13. The method of claim 12, wherein the method is such that a specific gene amplification reaction after performing a reverse transcription of an RNA template using the primer of claim 1 is carried out in a one-step process.
14. A composition for amplifying a gene used in a polymerase chain reaction method, a real-time polymerase chain reaction method or an isothermal amplification method for gene amplification of DNA and RNA, the composition comprising the primer of claim 1 as an active ingredient.
15. The composition of claim 14, further comprising one or more polymerases selected from the group consisting of Taq polymerase having 5′.fwdarw.3′ exonuclease activity, HotStart Taq polymerase, PFU polymerase, and Klenow polymerase having 5′.fwdarw.3′ exonuclease (-) and 3′.fwdarw.5′ exonuclease (-) activity.
16. A kit for amplifying a specific gene for DNA and RNA of an infectious disease, a hereditary disease, drug tolerance, drug resistance, and a susceptible test sample, the kit comprising the primer of claim 1 as an active ingredient.
Description
DESCRIPTION OF DRAWINGS
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MODES OF THE INVENTION
[0053] Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1: Analysis of Effects of the Linker of the Bubble Portion 1 of the Primer (CP) of the Present Invention
[0054] DNA template for HIV tat amplification was prepared in which HIV tat plasmid DNA was prepared by a gene synthesis of HIV-1 isolate 10BR_PE064 (GI: 672918720, 5281-5700 bp) to plasmid DNA.
[0055] In the primer (CP) of the present invention, the forward primer and reverse primer for the HIV tat gene of the conventional method (Nucleic Acids Research, 36:20, 2008) were designed as target regions, the complementary sequence thereto was positioned at the 5′-terminal thereof, and the complementary sequence included 7 to 8 mis-matched sequences. The inosine linker was designed as SEQ ID NOS: 3 to 10 at the bubble portion 1 between the target region and the complementary sequence. SEQ ID NO: 3 was a primer including no linker in the bubble portion 1, and SEQ ID NOS: 4 to 10, respectively, were designed to include 1 (Ix1a, Ix1b, Ix1c), 3 (Ix3), 5 (Ix5), 7 (Ix7), and 9 (Ix9) inosine linkers.
[0056] The control group was used a mixture in which SEQ ID NO 1 was used as a forward primer and SEQ ID NO 2 was used as a reverse primer in the prepared primer. The experimental group was prepared in a mixture in which the primers (CP) of the present invention of SEQ ID NOS: 3 to 10 were used as a forward primer and SEQ ID NO 2 was used as a reverse primer.
[0057] The mixture for the nucleic acid amplification reaction using the primers was prepared in a reaction mixture including 3.0 mM MgCl.sub.2, 0.2 mM dNTPs (NEB), 1.75 U Taq DNA polymerase, 10 ng Human genomic DNA, and 0.5 M primer set. The nucleic acid amplification reaction was performed in that 45 ul of the reaction mixture and 5 ul of the template DNA in 69, 6.9 and 0.69 fg/ul, respectively, were added, and the initial denaturation step at 94° C. for 10 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 55° C. for 30 seconds, and the extension step at 72° C. for 2 minutes were carried out for 40 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
[0058] As a result, as illustrated in
Example 2: Analysis of Effects According to the Number of Mis-Matched Sequences on a Bubble Portion 2 of the Primer (CP) of the Present Invention
[0059] HIV tat plasmid DNA was used as a template. The primer (CP) of the present invention included one inosine on the bubble portion 1, and then 0, 3, 8, and 12 numbers of mis-matched sequences were included on the bubble portion 2, thereby preparing SEQ ID NOS: 11, 12, 4, and 13.
[0060] The control group was used a mixture in which SEQ ID NO: 1 was used as a forward primer and SEQ ID NO: 2 was used as a reverse primer in the prepared primer. The experimental group was prepared in a mixture in which the primers (CP) of the present invention of SEQ ID NOS: 11, 12, 4, and 13 were used as a forward primer and SEQ ID NO: 2 was used as a reverse primer.
[0061] The mixture for the nucleic acid amplification reaction using the primers was prepared in a reaction mixture including 3.0 mM MgCl.sub.2, 0.2 mM dNTPs, 1.75 U Taq DNA polymerase, 10 ng Human genomic DNA, and 0.5 μM primer set. The nucleic acid amplification reaction was performed in that 45 ul of the mixture and 5 ul of the template DNA in 69, 6.9 and 0.69 fg/ul, respectively, were added, and the initial denaturation step at 94° C. for 10 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 55° C. for 30 seconds, and the extension step at 72° C. for 2 minutes were carried out for 40 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
[0062] M0 illustrated in
[0063] M8 illustrated in
Example 3: Analysis of Effects According to the Number of Bubbles of the Primer (CP) of the Present Invention
[0064] The primer (CP) of the present invention includes inosine on a bubble portion 1 and mis-matched sequence on a bubble portion 2. The bubble portion 1 formed one bubble, and the bubble portion 2 formed three bubbles.
[0065] SEQ ID NO: 13 produced totally two bubbles, one on the bubble portion 1 and one on the bubble portion 2. SEQ ID NO: 14 produced one bubble on the bubble portion 1 and two bubbles on the bubble portion 2. SEQ ID NO: 15 produced one bubble on the bubble portion 1 and three bubbles on the bubble portion 2. Using the primer (CP) of the present invention as a forward primer and using SEQ ID NO: 2 as a reverse primer, the mixture for the nucleic acid amplification reaction was prepared in a reaction mixture including 3.0 mM MgCl.sub.2, 0.2 mM dNTPs, 1.75 U Taq DNA polymerase, 10 ng Human genomic DNA, and 0.5 μM primer set.
[0066] The nucleic acid amplification reaction was performed in that 45 ul of the reaction mixture and 5 ul of the template DNA in 69, 6.9 and 0.69 fg/ul, respectively, were added, and the initial denaturation step at 94° C. for 10 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 45° C. for 30 seconds, and the extension step at 72° C. for 2 minutes were carried out for 40 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
[0067] As a result of using the primer (CP) of the present invention in which the number of bubbles is 2 in each A line, the number of bubbles is 3 in each B line, and the number of bubbles is 4 in each C line as illustrated in
Example 4: Analysis of the Amplification Reaction of the Primer (CP) of the Present Invention by Type of DNA Polymerase
[0068] In order to confirm the amplification reaction of the primer (CP) of the present invention according to the DNA polymerase used in the nucleic acid amplification reaction, the primer (CP) of the present invention of SEQ ID NO: 4 was used as a forward primer, and SEQ ID NO: 2 was used as a reverse primer. The amplification reaction was analyzed by Taq DNA polymerase, HotStart Taq DNA polymerase, Vent DNA polymerase, and Pfu DNA polymerase.
[0069] In the nucleic acid amplification reaction as illustrated in
[0070] In the nucleic acid amplification reaction as illustrated in
[0071] In the nucleic acid amplification reaction as illustrated in
[0072] In the nucleic acid amplification reaction as illustrated in
[0073] The nucleic acid amplification reaction was performed in that 45 ul of the reaction mixture and 5 ul of the template DNA in 69, 6.9 and 0.69 fg/ul, respectively, were added, and the initial denaturation step at 94° C. for 10 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 45° C. for 30 seconds, and the extension step at 72° C. for 2 minutes were carried out for 40 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
[0074] As a result of the Taq DNA polymerase as illustrated in
[0075] As a result of the Hotstart Taq DNA polymerase as illustrated in
[0076] As a result of the Pfu DNA polymerase as illustrated in
[0077] As a result of the Vent DNA polymerase as illustrated in
Example 5: Analysis of Amplification Reaction According to the Application of Each or Pair of the Forward and Reverse Primers of the Primer (CP) of the Present Invention
[0078] As illustrated in
[0079] The primer (CP) of the present invention includes one inosine in the bubble portion 1 and four mis-matched sequences in the bubble portion 2 to produce the primer (CP) of the present invention having SEQ ID NO: 18 and SEQ ID NO: 19 for SEQ ID NO: 16 and SEQ ID NO: 17.
[0080] Using the primer (CP) of the present invention, the nucleic acid amplification reaction was carried out in which SEQ ID NO: 18 of the primer (CP) of the present invention was used as a forward primer and a conventional SEQ ID NO: 17 primer was used as a reverse primer in A_F line as illustrated in
[0081] The mixture of the nucleic acid amplification reaction for the primer set was prepared in a reaction mixture including 2.5 mM MgCl.sub.2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, and 0.5 μM primer set.
[0082] The reaction mixture and 100, 10, and 1 ng, respectively, of human genomic DNA (Promega, G3041) were added and then total 50 ul of reaction product was subject to the nucleic acid amplification reaction in which the initial denaturation step at 94° C. for 2 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 60° C. for 30 seconds, and the extension step at 72° C. for 45 seconds were carried out for 40 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
[0083] As a result according to the nucleic acid amplification reaction, a single-band amplification products of the A_F, A_R, and A_F & R lines using the primer (CP) of the present invention were exhibited, and there or more bands, as the amplification products, in the Con line using the conventional primer were exhibited. The amplification product for the beta actin gene was a single amplification product corresponding to 653 bp, and the result according to the primer (CP) of the present invention of
[0084] As illustrated in
[0085] The mixture of the nucleic acid amplification reaction for the primer set was prepared in a reaction mixture including 2.0 mM MgCl.sub.2, 0.2 mM dNTPs, 1 U Taq DNA polymerase, and 0.5 μM primer set.
[0086] The reaction mixture and 1000, 100, 10, and 1 pg, respectively, of Rat genomic DNA (Clonetech, Cat. No. 636404) were added and then total 25 ul of reaction product was subject to the nucleic acid amplification reaction in which the initial denaturation step at 94° C. for 5 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 55° C. for 30 seconds, and the extension step at 72° C. for 30 seconds were carried out for 35 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
[0087] The line 1 illustrated in
Example 6: Verification of Amplification Efficiency of RT-PCR Using the Primer (CP) of the Present Invention
[0088] RT-PCR was performed using a conventional primer (Nucleic Acids Research, 36:20, 2008) and the primer (CP) of the present invention to amplify the beta actin gene in human total RNA (Stratagene, Cat. No. 750500). In order to synthesize RNA into cDNA as the first step of RT-PCR, SEQ ID NO: 17 corresponding to the reverse primer of the conventional primer set was used, and, cDNA was synthesized using SEQ ID NO: 19 with 4 mis-matched sequences, SEQ ID NO: 27 with 6 mis-matched sequences, and SEQ ID NO: 29 with 8 mis-matched sequences, respectively, in the bubble portion 2 of the primer (CP) of the present invention.
[0089] For cDNA synthesis, a mixture was prepared to include 1× buffer (50 mM Tris-HCl, pH 8.3, 3 mM MgCl.sub.2, 10 mM DTT, 75 mM KCl), 2 mM dNTPs, 1 M primer, 200 U M-MLV RTase, and Human total RNA 40, 4, 0.4 ng.
[0090] The mixture for cDNA synthesis reaction reacted at 45° C., 55° C., and 65° C., respectively, for 60 minutes, and then the M-MLV RTase was inactivated at 94° C. for 5 minutes. The nucleic acid amplification reaction was carried out, in which the cDNA reaction solution was used as a template, and SEQ ID NO: 16 and SEQ ID NO: 17 were used as a primer set for the reaction solution synthesized in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 were used as a primer set for the reaction solution synthesized in SEQ ID NO: 19, SEQ ID NO: 26 and SEQ ID NO: 27 were used as a primer set for the reaction solution synthesized in SEQ ID NO: 27, and SEQ ID NO: 28 and SEQ ID NO: 29 were used as a primer set for the reaction solution synthesized in SEQ ID NO: 29, respectively.
[0091] The mixture of the nucleic acid amplification reaction for the primer set was prepared in a reaction mixture including 2.5 mM MgCl.sub.2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, and 0.5 M primer set.
[0092] The reaction mixture and each 5 ul of cDNA reaction solution (2, 0.2, and 0.02 ng) of each primer for Human total RNA were added and then total 50 ul of the reaction product was subject to the nucleic acid amplification reaction in which the initial denaturation step at 94° C. for 2 minutes, the denaturation step at 94° C. for 30 seconds, the annealing step at 60° C. for 30 seconds, and the extension step at 72° C. for 45 seconds were carried out for 40 cycles and the final extension step were carried out at 72° C. for 7 minutes. 5 ul of the amplification product was subjected to electrophoresis analysis on 2% agarose gel including gel green dye.
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