KR20130118602A - Polynucleotide and use thereof - Google Patents

Abstract

PURPOSE: A polynucleotide is provided to efficiently amplify a target nucleic acid. CONSTITUTION: A polynucleotide contains two or more regions which are complementary to a target nucleic acid. A first complementary region (1) is one or more continuous nucleotides from 3' end, which is complementary to a target nucleic acid (4). One or more secondary complementary regions (2) are one or more continuous nucleotides in a 5' end direction of the first complementary region, which is complementary to the target nucleic acid. The first complementary region is hybridized in a 3' end direction of the second complementary region. A composition for amplifying the target nucleic acid contains the polynucleotide and the target nucleic acid.

Description

Polynucleotides and uses thereof < RTI ID = 0.0 > (Polynucleotide and use thereof)

Polynucleotides comprising two or more complementary regions that are complementary to the target nucleic acid and are in reverse configuration, and uses thereof.

Amplification of the nucleic acid comprises extending the nucleotide sequence from the 3'-end of the primer in the presence of the nucleic acid polymerase. The primers include sequences complementary to the target nucleic acid. For extension of the sequence, the primer needs to be stably hybridized specifically with the target nucleic acid. The stability of the nucleic acid hybridization product is known to be proportional to the length of the complementary sequence. On the other hand, as the length of the primer becomes longer, the length of the amplified target nucleic acid becomes shorter.

Even in the prior art, there is a demand for a polynucleotide capable of increasing the length of a target nucleic acid that is specifically and stably bound to a target nucleic acid while being amplified.

One aspect provides polynucleotides capable of efficiently amplifying a target nucleic acid.

Another aspect provides a composition comprising a polynucleotide capable of efficiently amplifying a target nucleic acid.

Another aspect provides a kit comprising a polynucleotide capable of efficiently amplifying a target nucleic acid.

Another aspect provides a method for efficiently producing complementary nucleotide sequences to a target nucleic acid.

One aspect is a polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one contiguous nucleotide from the 3'-terminus is complementary to the target nucleic acid, Wherein the at least one consecutive nucleotide in the 5 'direction of the complementary region is complementary to the target nucleic acid and the first complementary region hybridizes to the 5' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid .

In the polynucleotide, the second complementary region may be 1 to 3, for example, 1, 2, or 3. Also, the second complementary region may be one in which at least one contiguous nucleotide comprising the 5 'terminal nucleotide from the 5' terminus is complementary to the target nucleic acid. For example 1 to 50 nt such as 1-40, 1-30, 1-20, 1-10, 2-40, 2-30, 2-20, 2-10, 3-40, 3- 30, 3-20, or 3-10 nt. The second complementary region may comprise DNA, RNA, or a nucleotide analogue, such as PNA and LNA. The second complementary region may be such that at least three consecutive nucleotides are complementary to the target nucleic acid. For example, consecutive nucleotides of 3 to 50 nt, 3 to 40 nt, 3 to 30 nt, 3 to 20 nt, 3 to 10 nt, or 4 to 16 nt may be complementary to the target nucleic acid.

In the polynucleotide, the first complementary region may consist of two or more consecutive nucleotides complementary to the target nucleic acid. For example, a sequence of 2 to 50 nt, 2 to 40 nt, 2 to 30 nt, 2 to 20 nt, 2 to 10 nt, 3 to 50 nt, 4 to 40 nt, 3 to 30 nt, 3 to 20 nt, 3 to 10 nt, The nucleotide may be complementary to the target nucleic acid.

In the polynucleotide, the first complementary region and the second complementary region may be separated by one or more nucleotides. The first and second complementary regions may be spaced apart by the linker. The linker may be a nucleic acid, a nucleic acid variant, a protein, a sugar, or a lipid molecule. The nucleic acid variant may be one comprising PNA, or LNA. For example, the linker may comprise a nucleic acid comprising 0 to 50 nt. The length of the linker can be, for example, from 2 to 50 nt, from 2 to 40 nt, from 2 to 30 nt, from 2 to 20 nt, from 2 to 10 nt, from 3 to 50 nt, from 4 to 40 nt, from 3 to 30 nt, 4 to 10 nt. In this case, the linker may include a primer binding site, a restriction enzyme recognition site, or a probe binding site.

In such polynucleotides, the first complementary region may be complementary to one or more contiguous nucleotides comprising the 3'-terminal nucleotide of the target nucleic acid, including the 3'-terminal nucleotide. For example, two or more consecutive nucleotides containing 3'-terminal nucleotides from the 3'-end of the target nucleic acid may be complementary to the target nucleic acid. For example, a sequence of 2 to 50 nt, 2 to 40 nt, 2 to 30 nt, 2 to 20 nt, 2 to 10 nt, 3 to 50 nt, 4 to 40 nt, 3 to 30 nt, 3 to 20 nt, 3 to 10 nt, The nucleotide may be complementary to the target nucleic acid.

In the polynucleotide, the second complementary region may be complementary to one or more contiguous nucleotides from the 5'-end of the target nucleic acid. For example, the second complementary region may be complementary to one or more contiguous nucleotides comprising a 5'-terminal nucleotide of the target nucleic acid and a 5'-terminal nucleotide. In this case, the second complementary region may be complementary to two or more consecutive nucleotides. For example, a sequence of 2 to 50 nt, 2 to 40 nt, 2 to 30 nt, 2 to 20 nt, 2 to 10 nt, 3 to 50 nt, 4 to 40 nt, 3 to 30 nt, 3 to 20 nt, 3 to 10 nt, The nucleotide may be complementary to the target nucleic acid.

The polynucleotide may be DNA or RNA. In addition, the polynucleotide may comprise a nucleotide analogue such as PNA and LNA. The polynucleotide may, for example, comprise a nucleotide analog, for example, PNA and LNA in a second complementary region. However, the nucleotide analogs, such as PNA and LNA, may be included in the first complementary region as well as the second complementary region. The polynucleotide may be a single strand. The polynucleotide may have a length of 7 nt to 200 nt, 7 nt to 180 nt, 7 nt to 150 nt, 7 nt to 130 nt, 7 nt to 100 nt, 7 nt to 80 nt, 7 nt to 50 nt, 30 nt, 7 nt to 20 nt, 7 nt to 15 nt, or 10 nt to 40 nt.

The target nucleic acid may be DNA, RNA or a chimera of DNA and RNA. The target nucleic acid may be single-stranded or double-stranded. Wherein the target nucleic acid has a length of 20 nt to 200 nt, 20 nt to 180 nt, 20 nt to 150 nt, 20 nt to 130 nt, 20 nt to 100 nt, 20 nt to 80 nt, 20 nt to 50 nt, 20 nt To 30 nt, or from 20 nt to 40 nt. The target nucleic acid may be small RNA. Small RNA may be, for example, microRNA, siRNA or tRNA. In addition, the target nucleic acid is RNA having a nucleotide of 200 nt or more, wherein a region in which the GC content of the continuous 30 nt sequence is 30% or 80% or more exists, a nucleotide sequence complementary to each other in the molecule is continuously present in 5 nt or more An RNA capable of forming a secondary structure in a molecule, an RNA having five or more consecutive nt's in the same nt, or a combination thereof.

The first complementary region and the second complementary region of the polynucleotide may hybridize to the target nucleic acid by being separated from each other by at least 1nt when hybridized to the target nucleic acid. For example, 1-20 nt, 1-10 nt, 1-5 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt or 11 nt apart.

The polynucleotide can act as a primer in template-dependent nucleic acid synthesis. Thus, the polynucleotide may be used as a primer. The polynucleotide can be used as a probe for confirming the presence of a target nucleic acid in a sample.

Another aspect is a polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one contiguous nucleotide from the 3'-terminus is complementary to the target nucleic acid, Wherein the at least one consecutive nucleotide in the 5 'direction of the complementary region is complementary to the target nucleic acid and the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid; And a target nucleic acid. ≪ / RTI >

In the composition, the polynucleotide is as described above. The target nucleic acid is as described above.

The composition is a composition for amplifying a target nucleic acid. The amplification may be a known method for nucleic acid amplification. The amplification may be, for example, DNA amplification or RNA amplification. The amplification method may be one that requires thermal cycling or is performed at isothermal. The amplification methods include polymerase chain reaction (PCR), NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), strand displacement amplification (SDA), rolling circle amplification amplification: RCA). The amplification method may also include an RNA amplification method. For example, reverse transcription (RT) or reverse transcription-PCR. Amplification may be to increase the number of copies of the target nucleic acid sequence or its complementary sequence. "PCR" may be a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase.

Thus, the composition may further comprise known materials required for amplification of the target nucleic acid. For example, the composition may further comprise a nucleic acid polymerase, a buffer necessary for its activity, cofactors, and / or a substrate. The nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, a reverse transcriptase, or a combination thereof. Reverse transcription may be the synthesis of DNA strands complementary to RNA sequences using RNA as a template. The nucleic acid polymerase may have a strand displacement activity. For example, it may be one or more reverse transcriptase derived from retroviruses such as HIV, MMLV, and AMV. The nucleic acid polymerase may not have 3 '->5' exonuclease activity. The composition may comprise a material necessary for reverse transcription or PCR amplification.

Another aspect is a polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one contiguous nucleotide from the 3'-terminus is complementary to the target nucleic acid, Wherein the at least one consecutive nucleotide in the 5 'direction of the complementary region is complementary to the target nucleic acid and the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid; And a kit for amplifying a target nucleic acid comprising a target nucleic acid.

In the kit, the polynucleotide is as described above. The target nucleic acid is as described above.

The kit is for amplifying the target nucleic acid. The amplification may be a known method for nucleic acid amplification. The amplification may be, for example, DNA amplification or RNA amplification. The amplification method may be one that requires thermal cycling or is performed at isothermal. The amplification methods include polymerase chain reaction (PCR), NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), strand displacement amplification (SDA), rolling circle amplification amplification: RCA). The amplification method may also include an RNA amplification method. For example, reverse transcription (RT) or reverse transcription-PCR. Amplification may be to increase the number of copies of the target nucleic acid sequence or its complementary sequence. "PCR" may be a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase.

Thus, the kit may further comprise known materials required for amplification of the target nucleic acid. For example, the kit may further comprise a nucleic acid polymerase, a buffer required for its activity, cofactors, and / or a substrate. The nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, a reverse transcriptase, or a combination thereof. Reverse transcription may be the synthesis of DNA strands complementary to RNA sequences using RNA as a template. The nucleic acid polymerase may have a strand displacement activity. For example, it may be a reverse transcriptase derived from retroviruses such as HIV, MMLV, and AMV. The nucleic acid polymerase may not have 3 '->5' exonuclease activity. The kit may comprise a material necessary for reverse transcription or PCR amplification. The kit may further include instructions for use to amplify the target nucleic acid.

Another aspect is a polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one contiguous nucleotide from the 3'-terminus is complementary to the target nucleic acid, Wherein one or more consecutive nucleotides in the 5 'direction of the complementary region are complementary to the target nucleic acid, and wherein the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid, ; ≪ / RTI > And incubating the hybridization product in the presence of a nucleic acid polymerase to elongate a nucleotide sequence complementary to the target nucleic acid from the 3'-end of the polynucleotide to produce a nucleotide sequence complementary to the target nucleic acid ≪ / RTI >

The method comprises: a polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one contiguous nucleotide from the 3'-terminus is complementary to the target nucleic acid, Wherein one or more consecutive nucleotides in the 5 'direction of the 1 < st > complementary region are complementary to the target nucleic acid, and wherein the first complementary region hybridizes to the 3'direction of the second complementary region based on the nucleotide sequence of the target nucleic acid, And hybridizing the nucleic acid.

The hybridization may be performed by known methods. For example, by incubating the polynucleotide with the target nucleic acid in a buffer known to be appropriate for the hybridization of the nucleic acid. The hybridization may be carried out at an appropriate temperature, for example, 0 ° C to 25 ° C, or 4 ° C. The temperature can be appropriately selected according to the sequence and length of the polynucleotide and the target nucleic acid to be selected. The hybridization may be for an appropriate period of time, for example from 1 hour to 12 hours (overnight). In the hybridization step, the polynucleotide and the target nucleic acid used are as described above.

The method comprises incubating the hybridization product in the presence of a nucleic acid polymerase to elongate a nucleotide sequence complementary to the target nucleic acid from the 3'-end of the polynucleotide.

The incubation may be carried out under conditions suitable for the activity of the nucleic acid polymerase. The incubation can be carried out in the presence of a nucleic acid polymerase, a buffer necessary for its activity, a cofactor, and a substrate. The nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, a reverse transcriptase, or a combination thereof. Reverse transcription may be the synthesis of DNA strands complementary to RNA sequences using RNA as a template. The nucleic acid polymerase may have a strand displacement activity. For example, it may be a reverse transcriptase derived from retroviruses such as HIV, MMLV, and AMV. The nucleic acid polymerase may not have 3 '-> 5' exonuclease activity. For example, the incubation may include performing under conditions that include materials necessary for reverse transcription or PCR amplification.

By the incubation, the nucleotide sequence complementary to the target nucleic acid can be extended from the 3'-end of the polynucleotide. The extension comprises extending the nucleic acid polymerase from the 3'-end of the polynucleotide while substituting the 5'-end of the polynucleotide hybridized with the target nucleic acid.

The method may further comprise determining the presence of a produced product, i. E., A nucleotide sequence complementary to the target nucleic acid. Determining that the target nucleic acid is present in the sample when the product is present, and determining that the target nucleic acid is not present in the sample if the product is not present can do.

The method may further comprise amplifying the nucleic acid using the produced product, that is, the nucleotide sequence complementary to the target nucleic acid as a template. The amplification can be performed by a known method. The amplification method is as described above.

The method may further comprise determining the presence of a nucleotide sequence complementary to the product produced, i.e., the target nucleic acid, as amplified. Determining that the target nucleic acid is present in the sample if the amplification product is present, and, if the amplification product is not present, determining that the target nucleic acid is not present in the sample have.

The method comprising a polynucleotide comprising at least two complementary regions complementary to a target nucleic acid, the polynucleotide comprising at least one nucleotide sequence that is mismatched with a target nucleic acid in a first complementary region, wherein the first complementary region comprises a 3 & Wherein at least one contiguous nucleotide is complementary to the target nucleic acid and at least one second complementary region is one in which at least one consecutive nucleotide in the 5'direction of the first complementary region is complementary to the target nucleic acid and the first complementary region is a nucleotide of the target nucleic acid And hybridizing the target nucleic acid with a polynucleotide that is located on the 5'-direction side of the second complementary region based on the sequence. The mismatch may be a 1 to 4nt mismatch, for example, a 1, 2, 3, or 4nt mismatch. The mismatch may be one nucleotide mismatch located at the first, second, third, fourth, fifth, or sixth position from the 3'-end of the first complementary region. The method can include comparing the elongated product obtained by the method with the polynucleotide comprising a mismatch and the polynucleotide not comprising a mismatch.

In addition, it may include determining whether a mutation is present in the target nucleic acid based on the result of the comparison. For example, when a product extending from the polynucleotide including a mismatch is obtained but no product extending from the polynucleotide not containing mismatch is obtained, the nucleotide of the target nucleic acid corresponding to the mismatch is And judging that there is a mutation. Conversely, when a product extending from the polynucleotide including a mismatch is not obtained but an extension extending from the polynucleotide not containing mismatch is obtained, the nucleotide of the target nucleic acid corresponding to the mismatch is mutated It may be determined that there is none.

Brief Description of the Drawings Figure 1 shows a method of producing a polynucleotide according to one aspect and a nucleotide sequence complementary to a target nucleic acid. 1, one or more consecutive nucleotides from the 3'-terminus of the first complementary region 1 are complementary to the target nucleic acid 4 and one or more second complementary regions 2 are complementary to the first complementary region 2 1 of the second complementary region 2 is complementary to the target nucleic acid 4 and one or more consecutive nucleotides in the 5'-direction of the second complementary region 2 are complementary to the target nucleic acid 4, 'Direction. Because the first and second complementary regions are complementary to the target nucleic acid, they contribute synergistically to the hybridization, which can contribute synergistically to the stabilization of the hybridization product. Thus, the length of the first complementary region contributing to priming in the nucleic acid amplification reaction can be shortened without loss of amplification specificity, sensitivity and / or speed. In addition, when the 3'-terminal of the polynucleotide is extended by the nucleic acid polymerase (5), the target nucleic acid can be amplified. In Figure 1, the 3'-terminal extension (5) of the polynucleotide involves reverse transcription or DNA strand synthesis from a DNA strand. The first complementary region and the second complementary region hybridized to the target nucleic acid may be located apart by at least 1 nt of nucleotides.

According to one aspect, polynucleotides can be used to efficiently amplify a target nucleic acid.

According to a composition and kit according to one aspect, it can be used to efficiently amplify a target nucleic acid.

According to a method of producing a nucleotide sequence complementary to a target nucleic acid according to an aspect, it is possible to efficiently produce a nucleotide sequence complementary to a target nucleic acid.

Brief Description of the Drawings Figure 1 shows a method of producing a polynucleotide according to one aspect and a nucleotide sequence complementary to a target nucleic acid.
FIG. 2 is a graph showing the influence of the length of the first complementary region on the extension efficiency.
Figure 3 shows the effect of reaction temperature and primer concentration on the extension efficiency of the 3'-terminal of RCP by reverse transcription.
FIG. 4 shows the effect of the length of the second complementary region on the extension efficiency when the target nucleic acid is miR-3141.
FIG. 5 shows the effect of the length of the second complementary region on the extension efficiency when the target nucleic acid is miR-16.
FIGS. 6 to 8 are diagrams showing the effect of the first complementary region including the nucleotide corresponding to the target nucleic acid sequence on the extension efficiency. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One

In this embodiment, a polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is complementary to the target nucleic acid with at least one consecutive nucleotide from the 3'-terminus, Wherein one or more consecutive nucleotides in the 5 'direction of the first complementary region are complementary to the target nucleic acid, and the first complementary region is located in the 5' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid The sequence complementary to the target nucleic acid was extended from the 3'-end using a "reverse configuration primer," or "RC primer", or RCP. As a control, a conventional primer without reverse- Linear primer "or" linear primer ") was used. In the inverse primer, the first complementary region Quot; priming region ", the second complementary region is also referred to as an " adapter region "or" adapter ".

1. The effect of length of first complementary region on extension efficiency

In the inverse hybridization primer, extension reaction was performed while changing the length of the first complementary region from 3mer to 7mer. The extension reaction was a reverse transcription reaction.

As a reverse transcriptase, Superscript III ^™ (Invitrogen) was used. Reverse transcription was performed by incubating the reverse-phase primer and the RNA template at 42 ° C for 1 hour in 50 mM Tris-HCl (pH 8.3 at room temperature) containing 37.5 mM KCl, 3 mM MgCl ₂ , and 10 mM DTT. The RNA template was miR-16 RNA (SEQ ID NO: 9) to which 20 nt tag sequence was added at the 5'-end. Superscript III ^TM reverse transcriptase is a variant of Maloney murine leukemia virus (M-MLV) reverse transcriptase engineered to provide reduced thermal stability and reduced RNA H activity.

The resulting elongation product was amplified by PCR. PCR was performed by adding 50 nM of each primer and extension product to the 2xSYBR RT-PCR master mix (Exiqon) and thermocycling. Thermal circulation was performed 45 times at 95 캜 for 10 minutes, at 95 캜 for 15 seconds, and at 60 캜 for 1 minute. Next, melting curve analysis was performed at 5 measurements / 占 폚. The RT primers used were RCP-3mer (SEQ ID NO: 1), RCP-4mer (SEQ ID NO: 2), RCP-6mer (SEQ ID NO: 3), RCP-7mer Linear-4mer (SEQ ID NO: 6), Linear-6 (SEQ ID NO: 7), and Linear-7mer (SEQ ID NO: 8). The PCR primer used was a forward primer (SEQ ID NO: 10) and a reverse primer (SEQ ID NO: 11).

FIG. 2 is a graph showing the influence of the length of the first complementary region on the extension efficiency. As shown in FIG. 2, when the length of the first complementary region was 3-mer and 4-mer, the Cp value was significantly decreased when RCP was used as compared with the linear primer. When the length of the first complementary region was 4mer, the Cp value decreased by 6.3 as compared with the control.

2. Reaction temperature, and primer  Effect of concentration on elongation efficiency

As a result of (1), the target nucleic acid was amplified using RCP and the control primer in which the first complementary region was 6-mer, which showed a small difference in elongation efficiency between the control and the RCP.

SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the target RNA (miR-3141 RNA to which the 20 nt tag sequence was added at the 5'-terminus) were used for the RCP and the control primer, respectively. The primer concentrations were 1 nM, 10 nM, and 100 nM, respectively, and the reverse transcription temperature was 42 ° C, 50 ° C, and 55 ° C, respectively. In addition, the reverse transcription and PCR conditions are as described in 1.

Figure 3 shows the effect of reaction temperature and primer concentration on the extension efficiency of the 3'-terminal of RCP by reverse transcription. As shown in Fig. 3, when RCP was used in comparison with the control group, the Cp value decreased.

3. Effect of length of 2nd complementary region on extension efficiency

The effect of the length of the second complementary region on the extension efficiency was confirmed in transcription and PCR amplification using RCP.

The target RNA used was miR-16 (SEQ ID NO: 9) to which 20 nt tag sequence was added at the 5'-end and miR-3141 (SEQ ID NO: 14) to which 20 nt tag sequence was added at the 5'-end. The RCPs used were SEQ ID NOS: 15, 16, 17, and 16, respectively, to which 4, 8, 10, 12, 14 and 15 mers (second complementary region) complementary to the target RNA were added at the 5 ' 18, 19, and 20 were used. SEQ ID NOS: 21, 22, 23, 24, 25, and 25, respectively, to which 4, 8, 10, 12, 14 and 16 mers (second complementary region) complementary to the target RNA were added at the 5 ' 26 was used.

FIG. 4 shows the effect of the length of the second complementary region on the extension efficiency when the target nucleic acid is miR-3141. As shown in FIG. 4, when the length of the second complementary region was 10mer, the Cp value was the lowest.

FIG. 5 shows the effect of the length of the second complementary region on the extension efficiency when the target nucleic acid is miR-16. As shown in FIG. 5, when the length of the second complementary region was 12mer, the Cp value was the lowest.

Table 1 (miR-3141) and Table 2 (miR-16) show the characteristics of the second complementary region.

The length of the second complementary region order GC content (%) Basic Tm (° C) Salt concentration adjusted Tm (℃) NN Tm (占 폚) 4 CCTC 3 14 14 - 8 CCGCCCTC 7 30 30 18.73 10 ACCCGCCCTC 8 36 36 31.49 12 CCACCCGCCCTC 10 44 44 40.98 14 CTCCACCCGCCCTC
11 49.1 52.7 46.58

The length of the second complementary region order GC content (%) Basic Tm (° C) Salt concentration adjusted Tm (℃) NN Tm (占 폚) 4 TGCT 2 12 12 - 8 GTGCTGCT 5 26 26 8.98 10 ACGTGCTGCT 6 32 32 28.32 12 TTACGTGCTGCT 6 36 36 33.97 14 ATTTACGTGCTGCT 6 34.4 38 38.41 16 ATATTTACGTGCTGCT 6 38.3 43.2 41.29

NN in Tables 1 and 2 represents a nearest neighbor.

4. In the target nucleic acid sequence Mitch Matchin  Effect of first complementary region including nucleotide on extension efficiency

The effect on the extension efficiency was confirmed when a mismatch in nucleotide was included in the first complementary region of the target nucleic acid sequence in transcription using RCP and PCR amplification.

The primer is a sequence complementary to the target nucleic acid (hereinafter also referred to as "wild-type: WT") without the mismatch, and the length of the first complementary region is 0, 1, 2, 3, 4, , sequences corresponding to positions 1, 2, 3, 4, 5, and 6 (positions M1, M2, M3, M4, M5, and M6, respectively) from the 3'- end of miR-3141 are mismatched Respectively. When the length of the first complementary region is 6mer, the WT primer, M1, M2, M3, M4, M5 and M6 mismatch primers have sequences of SEQ ID NOS: 27, 28, 29, 30, 31, 32 and 33, respectively. MiR-3141 RNA of SEQ ID NO: 14 was used as the target nucleic acid.

When the length of the first complementary region is 4mer, the WT primers, M1, M2, M3, and M4 mismatch primers have sequences of SEQ ID NOS: 34, 35, 36, 37,

When the length of the first complementary region is 3mer, the WT primer, M1, M2, and M3 mismatch primers have sequences of SEQ ID NOs: 39, 40, 41, and 42, respectively.

When the length of the first complementary region is 2mer, the WT primer, M1 and M2 mismatch primers have sequences of SEQ ID NOS: 43, 44 and 45, respectively.

When the length of the first complementary region is 1mer, the WT primer and the M1 mismatch primer have sequences of SEQ ID NOS: 46 and 47, respectively.

When the length of the first complementary region is 0mer, the WT primers each have the sequence of SEQ ID NO: 48.

Primer concentrations of 10 nM, 100 nM and 1 uM were used. The other reverse transcription and PCR conditions are as described in 1.

FIGS. 6 to 8 are diagrams showing the effect of the first complementary region including the nucleotide corresponding to the target nucleic acid sequence on the extension efficiency. FIG. FIGS. 6 to 8 show the cases where the primer concentrations were 1 uM, 100 nM, and 10 nM, respectively. As shown in FIGS. 6 to 8, when the mismatch sequence was introduced into the first complementary region of the RCP, and when the mismatch sequence was not present, the Cp values were significantly different. 6 to 8, when the length of the first complementary region was 4-mer, the Cp value was further increased by 10.7-6.8 as compared with the WT primer. 6 to 8, the mismatch position was designated as M1, M2, M3, M4, M5, and M6 from the 3'-end in miR-3141 of SEQ ID NO: 14, respectively.

(M2) a (M1) g (M3) g (M3) g (M5) g (M5) a (M5) ggggcgggu ggaggcgggu ggaggggggu gg

6 to 8, it is shown that the presence of a mutation in the target nucleic acid can be confirmed by introducing a mismatch sequence into the first complementary region. Whether a mutation has been introduced into the target nucleic acid can be confirmed by comparing the amplification product obtained from the mismatch-introduced primer with the non-introduced primer.

<110> Samsung Electronics. Co. Ltd. <120> Polynucleotide and use thereof <130> PN096414 <160> 48 <170> Kopatentin 2.0 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> RCP-3mer <400> 1 ttacgtgctg ctaggtggta gccttgagga cacgc 35 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RCP-4mer <400> 2 ttacgtgctg ctaggtggta gccttgagga cacgcc 36 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> RCP-6mer <400> 3 ttacgtgctg ctaggtggta gccttgagga cacgccaa 38 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> RCP-7mer <400> 4 ttacgtgctg ctaggtggta gccttgagga cacgccaat 39 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Linear-3mer <400> 5 aggtggtagc cttgaggaca cgc 23 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Linear-4mer <400> 6 aggtggtagc cttgaggaca cgcc 24 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Linear-6mer <400> 7 aggtggtagc cttgaggaca cgccaa 26 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Linear-7mer <400> 8 aggtggtagc cttgaggaca cgccaat 27 <210> 9 <211> 42 <212> RNA <213> Artificial Sequence <220> <223> miR-16 RNA with 20 nt 5'-tag seqeuence <400> 9 cggugagguc uuugguucau uagcagcacg uaaauauugg cg 42 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward PCR primer <400> 10 cggtgaggtc tttggttcat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse PCR primer <400> 11 aggtggtagc cttgaggaca 20 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> RCP RT primer with 6mer 1st complementary region <400> 12 ccacccgccc tcaggtggta gccttgagga catcctcc 38 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Control RT primer <400> 13 aggtggtagc cttgaggaca cgatcgtcct cc 32 <210> 14 <211> 39 <212> RNA <213> Artificial Sequence <220> <223> miR-3141 with 5'-20nt tag sequence <400> 14 cggugagguc uuugguucau gagggcgggu ggaggagga 39 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 4mer adapter sequence <400> 15 cctcaggtgg tagccttgag gacatcct 28 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 8mer adapter sequence <400> 16 ccgccctcag gtggtagcct tgaggacatc ct 32 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 10mer adapter sequence <400> 17 acccgccctc aggtggtagc cttgaggaca tcct 34 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 12mer adapter sequence <400> 18 ccacccgccc tcaggtggta gccttgagga catcct 36 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 14mer adapter sequence <400> 19 ctccacccgc cctcaggtgg tagccttgag gacatcct 38 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 15mer adapter sequence <400> 20 cctccacccg ccctcaggtg gtagccttga ggacatcct 39 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 4mer adapter sequence complementary to          miR-16 <400> 21 tgctaggtgg tagccttgag gacacgcc 28 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 8mer adapter sequence complementary to          miR-16 <400> 22 gtgctgctag gtggtagcct tgaggacacg cc 32 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 10mer adapter sequence complementary to          miR-16 <400> 23 acgtgctgct aggtggtagc cttgaggaca cgcc 34 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 12mer adapter sequence complementary to          miR-16 <400> 24 ttacgtgctg ctaggtggta gccttgagga cacgcc 36 <210> 25 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 14mer adapter sequence complementary to          miR-16 <400> 25 atttacgtgc tgctaggtgg tagccttgag gacacgcc 38 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> RCP with 5 'terminal 16mer adapter sequence complementary to          miR-16 <400> 26 atatttacgt gctgctaggt ggtagccttg aggacacgcc 40 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region <400> 27 acccgccctc aggtggtagc cttgaggaca tcctcc 36 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region and M1 mismatch <400> 28 acccgccctc aggtggtagc cttgaggaca acctcc 36 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region and M2 mismatch <400> 29 acccgccctc aggtggtagc cttgaggaca tactcc 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region and M3 mismatch <400> 30 acccgccctc aggtggtagc cttgaggaca tcatcc 36 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region and M4 mismatch <400> 31 acccgccctc aggtggtagc cttgaggaca tccacc 36 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region and M5 mismatch <400> 32 acccgccctc aggtggtagc cttgaggaca tcctac 36 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 6mer 1st comlementary region and M6 mismatch <400> 33 acccgccctc aggtggtagc cttgaggaca tcctca 36 <210> 34 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 4mer 1st comlementary region <400> 34 acccgccctc aggtggtagc cttgaggaca tcct 34 <210> 35 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 4mer 1st comlementary region and M1 mismatch <400> 35 acccgccctc aggtggtagc cttgaggaca acct 34 <210> 36 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 4mer 1st comlementary region and M2 mismatch <400> 36 acccgccctc aggtggtagc cttgaggaca tact 34 <210> 37 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 4mer 1st comlementary region and M3 mismatch <400> 37 acccgccctc aggtggtagc cttgaggaca tcat 34 <210> 38 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 4mer 1st comlementary region and M4 mismatch <400> 38 acccgccctc aggtggtagc cttgaggaca tcca 34 <210> 39 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 3mer 1st comlementary region <400> 39 acccgccctc aggtggtagc cttgaggaca tcc 33 <210> 40 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 3mer 1st comlementary region and M1 mismatch <400> 40 acccgccctc aggtggtagc cttgaggaca acc 33 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 3mer 1st comlementary region and M2 mismatch <400> 41 acccgccctc aggtggtagc cttgaggaca tac 33 <210> 42 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 3mer 1st comlementary region and M3 mismatch <400> 42 acccgccctc aggtggtagc cttgaggaca tca 33 <210> 43 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 2mer 1st comlementary region <400> 43 acccgccctc aggtggtagc cttgaggaca tc 32 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 2mer 1st comlementary region and M1 mismatch <400> 44 acccgccctc aggtggtagc cttgaggaca ac 32 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RC primer with 2mer < 1 > <400> 45 acccgccctc aggtggtagc cttgaggaca aa 32 <210> 46 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 1mer 1st comlementary region <400> 46 acccgccctc aggtggtagc cttgaggaca t 31 <210> 47 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 1mer 1st comlementary region and M1 mismatch <400> 47 acccgccctc aggtggtagc cttgaggaca a 31 <210> 48 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RC primer with 0mer 1st comlementary region <400> 48 acccgccctc aggtggtagc cttgaggaca 30

Claims (20)

A polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one consecutive nucleotide from the 3'-terminus is complementary to the target nucleic acid, and at least one second complementary region comprises Wherein the at least one consecutive nucleotide in the 5 'direction is complementary to the target nucleic acid, and the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid. 3. The polynucleotide of claim 1, wherein the second complementary region is complementary to the target nucleic acid, wherein the at least one contiguous nucleotide comprises a 5'-terminal to 5'-terminal nucleotide. 2. The polynucleotide of claim 1, wherein the first complementary region is complementary to the target nucleic acid of 2 to 7 nt of consecutive nucleotides, including 3'-terminal nucleotides from the 3'-end. The polynucleotide of claim 1, wherein the first complementary region and the second complementary region are separated by a linker. 5. The polynucleotide of claim 4, wherein the linker is a nucleic acid, a nucleic acid variant, a protein, a sugar, or a lipid molecule. 6. The polynucleotide of claim 5, wherein the linker comprises a primer binding site, a restriction enzyme recognition site, or a probe binding site. The polynucleotide of claim 1, wherein the first complementary region is complementary to at least one contiguous nucleotide comprising a 3'-terminal nucleotide from the 3'-end of the target nucleic acid. The polynucleotide of claim 1, wherein the second complementary region is complementary to at least one contiguous nucleotide comprising the 5'-end of the target nucleic acid and a 5'-terminal nucleotide. The polynucleotide of claim 1, wherein the polynucleotide has a length of 7 nt to 100 nt. The polynucleotide of claim 1, wherein the target nucleic acid is 15 nt to 200 nt in length. The polynucleotide of claim 1, wherein the target nucleic acid is RNA. The polynucleotide of claim 1, wherein the target nucleic acid is a microRNA, siRNA, or tRNA. The polynucleotide of claim 1, wherein at least one of the first and second complementary regions comprises DNA, RNA, PNA or LNA. A polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one consecutive nucleotide from the 3'-terminus is complementary to the target nucleic acid, and at least one second complementary region comprises Wherein the at least one consecutive nucleotide in the 5 'direction is complementary to the target nucleic acid, and wherein the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid; And
A composition for amplifying a target nucleic acid, comprising a target nucleic acid. A polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one consecutive nucleotide from the 3'-terminus is complementary to the target nucleic acid, and at least one second complementary region comprises Wherein the at least one consecutive nucleotide in the 5 'direction is complementary to the target nucleic acid, and wherein the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid; And
A kit for amplifying a target nucleic acid comprising a target nucleic acid. A polynucleotide comprising two or more complementary regions complementary to a target nucleic acid, wherein the first complementary region is one wherein at least one consecutive nucleotide from the 3'-terminus is complementary to the target nucleic acid, and at least one second complementary region comprises Wherein the one or more consecutive nucleotides in the 5 'direction are complementary to the target nucleic acid, and wherein the first complementary region hybridizes to the 3' direction of the second complementary region based on the nucleotide sequence of the target nucleic acid step;
Incubating the hybridization product in the presence of a nucleic acid polymerase to extend the nucleotide sequence complementary to the target nucleic acid from the 3'-end of the polynucleotide to produce a nucleotide sequence complementary to the target nucleic acid . 17. The method of claim 16, wherein the nucleic acid polymerase is a strand-displaced nucleic acid polymerase. 19. The method of claim 17, wherein said stranded nucleic acid polymerase is at least one reverse transcriptase from HIV, MMLV, and AMV. 21. The method of claim 19, wherein the target nucleic acid is RNA. 17. The method of claim 16, wherein the target nucleic acid is a microRNA, siRNA, or tRNA.

Images