KR101922126B1 - Method for amplifying a target nucleic acid with reduced amplification bias and method for determining relative amount of a target nucleic acid in a sample - Google Patents

Abstract

A method for amplifying a target nucleic acid with reduced biases and a method for determining the relative amount of a target nucleic acid in a sample.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for amplifying a target nucleic acid having reduced bias and a method for determining a relative amount of a target nucleic acid in a sample.

More than one embodiment pertains to a method of amplifying a target nucleic acid and a method of determining the relative amount of a target nucleic acid in a sample.

Nucleic acid amplification means increasing the number of copies of the target sequence or its complementary sequence. Nucleic acid amplification is known in the art. Amplification of nucleic acids involves either a method requiring multiple cycles during amplification or a method performed at a single temperature. Examples of cycling techniques include those that require thermal cycling. Methods that require thermocycling include polymerase chain reaction (PCR). PCR is known in the art. PCR typically involves denaturing double stranded DNA to single stranded DNA by thermal degradation, annealing the primer to the single stranded DNA; And synthesizing a complementary strand from the primer.

An isothermal amplification method is a method in which a single temperature or a major aspect of the amplification process is performed at a single temperature. In contrast to PCR, in which the reaction product is heated to allow additional primers to bind in order to separate the double strands, the isothermal method relies on a strand displacing polymerase to separate the double strands and rescopy the template do. Isothermal methods can be distinguished by methods that rely on substitution of primers to initiate reiterative template copying and methods that rely on sequential reuse or novel synthesis of single primer molecules. Methods that rely on primer substitution include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA) and loop mediated amplification and loop mediated amplification (LAMP). Methods that rely on sequential reuse or novel synthesis of single primer molecules include those selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA).

On the other hand, in amplifying a short target nucleic acid sequence, an amplification bias may be generated depending on the type of the target nucleic acid. The amplification bias may vary depending on the copy number of the target nucleic acid or the base sequence, for example, the AT content or the GC content. For example, when a miRNA is amplified in a sample containing a plurality of microRNAs (miRNA), that is, a sample containing an miRNA library, the degree of amplification may vary depending on the sequence and copy number of the miRNA. miRNAs are generally known to have a length of about 18 to 25 nt, usually about 21 to 24 nt, and more than 1000 different sequences.

Thus, there is still a need for a method of amplifying a target nucleic acid from a sample containing a plurality of target nucleic acids, such as an miRNA library, in which the target nucleic acid is amplified with no or reduced amplification bias.

One embodiment is to provide a method of amplifying a target nucleic acid with a reduced amplification vise between the target nucleic acids.

Another embodiment provides a method for determining the relative amount of a target nucleic acid in a sample.

One aspect includes providing a sample comprising a plurality of types of target nucleic acids; Linking two or more of the target nucleic acids; And amplifying the ligated product. ≪ Desc / Clms Page number 2 >

The method includes providing a sample comprising a plurality of types of target nucleic acids. The sample may be of any form including a target nucleic acid. For example, the sample includes a biological sample or a chemical sample containing a target nucleic acid. The biological sample may be a sample derived from an organism and subjected to any additional process such as separation, purification and amplification from the organism itself as well as the separated organism itself. The separated organisms include, for example, at least one member selected from the group consisting of blood separated from the living body, urine, saliva, lymph, tissue and cells. In addition, a sample that has undergone further processing may be, for example, a serum, a nucleic acid isolated from an isolated organism, or a sample containing an amplified nucleic acid. The chemical sample may be one comprising a chemically synthesized target nucleic acid. The sample may be one comprising RNA or DNA. The sample may be a sample containing miRNA. The term " miRNA " can be a short RNA found in eukaryotic cells. miRNAs have important functions in cellular physiology and can act as post-translational regulators of gene expression. miRNAs may be isolated by methods based on TRizol / TRI-reagent solutions. miRNAs may be isolated according to known separation kits such as the mirVana isolation kit (Ambion) and RNeasy kit (Qiagen).

The target nucleic acid may have a length of 200 nt or less. The target nucleic acid can be, for example, about 15 to about 200 nt, about 15 to about 170 nt, about 15 to about 150 nt, about 15 to about 120 nt, about 15 to about 100 nt, about 15 to about 80 nt, From about 18 to about 80 nt, from about 18 to about 75 nt, from about 18 to about 70 nt, from about 18 to about 200 nt, from about 18 to about 170 nt, from about 18 to about 150 nt, from about 18 to about 150 nt, From about 18 nt to about 25 nt, from about 18 nt to about 25 nt, or from about 21 to about 25 nt. The target nucleic acid may, for example, comprise a size corresponding to pre-miRNA and miRNA. The target nucleic acid may be DNA or RNA. The RNA may be selected from the group consisting of tRNA, rRNA, mRNA and miRNA. The DNA may be a genomic DNA fragment or a cDNA prepared from RNA.

The term " plural kinds of target nucleic acids " means that two or more nucleic acid molecules having different primary sequences are present in one sample. For example, two or more miRNA molecules with different sequences are present in one sample.

The method comprises linking two or more of the target nucleic acids. The linkage may be accomplished by methods known in the art. For example, the linkage may be in the presence of one or more of RNA ligase and DNA ligase. Linking reaction conditions in the presence of one or more of RNA ligase and DNA ligase are known to those skilled in the art. The reaction conditions may include, for example, a nucleic acid having a 5'-phosphorylated terminus, a nucleic acid having a 3'-hydroxyl terminus, and a solution containing ATP. The RNA ligase includes, for example, T4 RNA ligase 1 or 2. DNA ligase includes T4 DNA ligase. The reaction may be carried out in a buffer suitable for the ligase reaction. The reaction temperature may be 35 占 폚 to 40 占 폚, for example, 37 占 폚.

The linkage may be by mediating an adapter sequence between the target nucleic acid and the target nucleic acid. The linkage may be such that an adapter sequence specific to the 3 'end is connected to the 3' end of the target nucleic acid and a target nucleic acid to which the adapter sequence specific to the 5 'end is connected to the 5' end of the target nucleic acid. The term " adapter sequence " refers to a nucleic acid sequence for linking to the 3 ' or 5 ' end of a target nucleic acid. The adapter sequence may be DNA or RNA. Adapter sequences may be single-stranded or double-stranded. The adapter sequences are those in which the GC content is between 20% and 80%, such as between 30% and 80%, between 40% and 80%, between 50% and 80%, between 60% and 80%, between 70% and 80% , 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 50%, or 40% to 70%. The adapter sequence can be from about 3 nt to about 100 nt in length. The adapter sequence may be from about 3 nt to about 70 nt, from about 3 nt to about 50 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3 nt to about 10 nt, from about 3 nt to about 7 nt From about 4 nt to about 20 nt, from about 4 nt to about 10 nt, or from about 4 nt to about 7 nt.

An adapter sequence specific for the '3' end or the 5 'end means that different adapter sequences are connected to the 3' and 5 'ends, respectively. This may be accomplished by selectively blocking or activating the reactivity of the 5'and 3'ends of the target nucleic acid and adapter sequences. For example, the 5'-end of a target nucleic acid (e. G., A miRNA or its complementary sequence cDNA) is phosphorylated and the 3'-end is blocked (e. G., Using a kinase to generate a 3'- -Phosphoryl group), and a 5 'terminal adapter sequence (A5) having an -OH group at the 3'-end is reacted in the presence of DNA or RNA ligase to form 5' end only at the 5'end of the target nucleic acid. You can connect the terminal adapter sequence. Here, the 5 ' end of the 5 ' terminal adapter sequence may be optionally phosphorylated or not. The resulting ligated product can be ligated with the 3'-terminal adapter sequence (A3) at the 5'-terminal phosphorylated site in the presence of DNA or RNA ligase to link the 3'-terminal adapter sequence to the 3'end of the linked product. Here, the 3 ' end of the 3 ' terminal adapter sequence may be optionally phosphorylated or not. The adapter sequence specific for the 3 'and 5' ends may be introduced first at the 5 'end, then at the 3' end, at the 3 'end first and then at the 5' end, Or introduced at the 5 ' end and the 3 ' end in the same reaction. The adapter sequence specific to the 5 'end and the adapter sequence specific to the 3' end may be different from each other or have the same nucleotide sequence.

Examples of the 5 'end and the 3' end introduced in the same reaction include a target nucleic acid in which the 5 'end is phosphorylated and the 3' end is the -OH group, the 5 'end is not phosphorylated, Terminal adapter sequence and 5 'end of the target nucleic acid are reacted in the presence of DNA or RNA ligase in the same reaction solution, and the 5' terminal and the 3 'terminal end of the 3' You can connect an adapter sequence.

The target sequence to which a specific adapter sequence is linked at one or more of the 5 'end and the 3' end, respectively, can also be linked to each other, as described above.

The method includes linking a primer sequence or a primer binding sequence to one or more of the 3 ' and 5 ' ends of the linked product to which the target nucleic acid and the target nucleic acid are linked. The linkage of the primer sequence or the primer binding sequence may be performed in the same manner as the linkage of the adapter sequence. At least one of the primer sequence and the primer binding sequence may be specifically linked to either the 3 'end or the 5' end. The term " primer binding sequence " includes a sequence complementary to the primer.

The term " primer sequence " refers to a nucleic acid sequence that serves as a starting point for nucleic acid polymerization. The primer sequence may bind to the target nucleic acid and have 3'-OH. The primer sequence may be DNA or RNA. The primer sequence may be a single strand. The primer sequence may be selected so that the GC content is between 20% and 80%, such as between 30% and 80%, between 40% and 80%, between 50% and 80%, between 60% and 80%, between 70% and 80% , 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 50%, or 40% to 70%. The primer sequence may be about 10 nt to about 100 nt in length. The primer sequence may be from about 10 nt to about 70 nt, from about 10 nt to about 50 nt, from about 10 nt to about 30 nt, from about 10 nt to about 20 nt, from about 15 nt to about 100 nt, from about 15 nt to about 70 nt From about 15 nt to about 50 nt, from about 15 nt to about 40 nt, or from about 15 nt to about 30 nt.

The method comprises amplifying the linked product. The term " amplification " refers to increasing the number of copies of the target sequence or its complementary sequence. Amplification may be accomplished by any method known in the art. Amplification of nucleic acids involves either a method requiring multiple cycles during amplification or a method performed at a single temperature. Examples of cycling techniques include those that require thermal cycling. Methods that require thermocycling include polymerase chain reaction (PCR). PCR is known in the art. PCR typically involves denaturing double stranded DNA to single stranded DNA by thermal degradation, annealing the primer to the single stranded DNA; And synthesizing a complementary strand from the primer. An isothermal amplification method is a method in which a single temperature or a major aspect of the amplification process is performed at a single temperature. In contrast to PCR, in which the reaction product is heated to allow additional primers to bind in order to separate the double strands, the isothermal method relies on a strand displacing polymerase to separate the double strands and rescopy the template do. Isothermal methods can be distinguished by methods that rely on substitution of primers to initiate reiterative template copying and methods that rely on sequential reuse or novel synthesis of single primer molecules. Methods that rely on primer substitution include helicase dependent amplification (HDA), exonuclease dependent amplification, recombinase polymerase amplification (RPA) and loop mediated amplification and loop mediated amplification (LAMP). Methods that rely on sequential reuse or novel synthesis of single primer molecules include those selected from the group consisting of strand displacement amplification (SDA) and nucleic acid based amplification (NASBA and TMA).

The amplification may be to amplify the linked product as an amplification product. That is, the primers can be selected so that all of the target nucleic acids contained in one linking product are contained in one amplification product. For example, in the case of PCR, the forward primer corresponds to the 5'end or upstream of the most upstream target nucleic acid, and the reverse primer is complementary to the 3'end of the most downstream target nucleic acid or downstream thereof have. The amplification may be performed using at least one of the primer sequence and the sequence complementary to the primer binding sequence as primers.

In one embodiment, the primer may be one that does not contain a sequence complementary to a target nucleic acid, e. G., MiRNA or its complementary cDNA. For PCR, the forward primer corresponds to the upstream of the 5 'end of the most upstream target nucleic acid and does not contain a target nucleic acid or a complementary sequence thereof, and the reverse primer contains the 3' terminal end of the most downstream target nucleic acid Is downstream complementary and may not contain a target nucleic acid or a complementary sequence thereof.

The method may further include, when the target nucleic acid is RNA, reverse transcribing the target nucleic acid to generate complementary DNA (cDNA) to the target nucleic acid. The reverse transcription may be performed before or after the connecting step. Reverse transcription is known in the art to generate RNA strands of DNA complement using reverse transcriptase.

The method may further comprise ligating the adapter sequence to one or more of the 3 'and 5' ends of the target nucleic acid prior to reverse transcription. The adapter sequence may be specifically linked at one or more of the 3 'and 5' ends.

The method may further comprise, after the reverse transcription, ligating the adapter sequence to one or more of the 3 'and 5' ends of the cDNA complementary to the target nucleic acid. The adapter sequence may be specifically linked at one or more of the 3 'and 5' ends. The connection of the adapter sequences is the same as described above.

Another aspect relates to a method of amplifying a target nucleic acid in a sample according to the method described above; Determining the amount of target nucleic acid from the resulting amplified product; And determining the relative amount of the target nucleic acid in the sample from the amount of the target nucleic acid.

The method comprises amplifying a target nucleic acid in a sample according to the method described above. This is the same as described above.

The method includes determining the amount of target nucleic acid from the resulting amplified product. The amount of target nucleic acid can be determined by methods known in the art. The amount of target nucleic acid can be determined by a real time amplification method. Real-time amplification methods include the Taqman method using fluorescent dyes and fluorescence quencher materials and the method using a fluorescent material that binds double-stranded DNA. The Taqman method is based on the amount of fluorescence detected during amplification, for example, by using a Taqman probe in which a fluorescent reporter substance is bound to the 5 'end and a fluorescent reporter is attached to the 3' end and a specific sequence is contained in each target nucleic acid The amount of the target nucleic acid can be determined in real time. As the primer is extended by a DNA polymerase having 5 '-> 3' exonuclease activity during amplification and new strands are synthesized, the 5 '-> 3' exonuclease activity is annealed to the template The probe is disassembled. Upon decomposition of the probe, the fluorescent dye is released, whereby the inhibition by the fluorescence inhibitor is released and the fluorescence can be emitted. Here, the fluorescent reporter material may be different depending on the target nucleic acid. Real-time amplification methods using Taqman probes are known, and any known method can be used.

In a manner using a fluorescent material that binds to double-stranded DNA, the DNA-binding fluorophore binds to all double-stranded DNA during PCR. An increase in the DNA product during the PCR allows the fluorescence intensity to be increased and the DNA concentration measured at each cycle to be quantified. The DNA-binding fluorescent material may be SYBR Green I. The amount of target nucleic acid can also be determined by amplification products after amplification, such as by hybridization methods or electrophoresis.

The method includes determining the relative amount of target nucleic acid in the sample from the amount of the target nucleic acid. The step of determining the relative amount of the target nucleic acid may be accomplished by standardizing the amount of target nucleic acid to a specific value, for example, a background value, or by determining a relative value by comparing with another standard value.

The relative amount of the determined target nucleic acid can be used to determine the association of the target nucleic acid with various physiological conditions, e.g., disease.

According to a method of amplifying a target nucleic acid according to one embodiment, a target nucleic acid can be amplified from a sample comprising a plurality of target nucleic acids with a reduced amplification vise between the target nucleic acids. Therefore, the profile of the target nucleic acid in the sample can be more accurately estimated from the amount of the amplification product.

By the method of determining the relative amount of the target nucleic acid in the sample according to another embodiment, the relative amount of the target nucleic acid in the sample can be efficiently determined.

Fig. 1 is a diagram showing the results of electrophoresis and analysis of amplification products.

Specific examples of 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 1 : Amplification Vaisa Reduced  Method of amplifying target nucleic acid

1. Individual miRNA Complementary to cDNA on adapter  Sequence and primer  Preparation of the First Linked Sequence with Sequence Linked

15 miRNAs with different GC content were selected as target nucleic acids to be amplified. In order to simulate the sequences in which the adapter sequences and the primer sequences were sequentially connected to the 3 'and 5' ends of these target nucleic acids, adapter sequences and primer sequences were inserted at the 3 'end and the 5' end, respectively, Sequentially combined single stranded DNA (hereinafter referred to as "first linked product") was synthesized.

Specifically, a 3 'end adapter sequence (hereinafter referred to as' A3') was attached to the 3 'end of DNA (cDNA) complementary to each miRNA and a 5' end adapter sequence (hereinafter referred to as' A5 ' (Bioneer Co., Ltd., Korea) was synthesized by attaching 3 'primer sequence (P3) to the 3' terminal adapter sequence and 5 'primer sequence (P5) attached to the 5' terminal adapter sequence. . The general structure of each thus synthesized first linkage product is the cDNA-A3-P3 of P5-A5-miRNA. Considered, P5 and P3 are primer binding sites. The sequences of P5-A5 and A3-P3 are as follows.

P5-A5: 5'-TGAGTTCTACGGTACCTCTAAGC-3 '(SEQ ID NO: 16)

A3-P3: 5'-AGGCATAAGCTGTTAGCTCAGAAT-3 '(SEQ ID NO: 17)

As the amplification primer, a reverse primer 5'-ATTCTGAGCTAACAGCTTATGCCT-3 '(SEQ ID NO: 18) complementary to the P5-A5 sequence and A3-P3 was used.

The adapter sequence and the primer sequence belong to the GC content of 20% to 80%, and the resultant cDNA product is linked to the cDNA so that the total GC content is changed in the range of 20% to 80%.

The sequences of the 15 miRNAs used are shown in Table 1.

turn designation SEQ ID NO: GC content Tm Length One hsa-mir-95 SEQ ID NO: 1 36.4 60 22 2 hsa-mir-7 SEQ ID NO: 2 34.8 62 23 3 hsa-mir-1 SEQ ID NO: 3 27.3 56 22 4 hsa-mir-9 SEQ ID NO: 4 34.8 62 23 5 hsa-mir-16 SEQ ID NO: 5 45.5 64 22 6 hsa-mir-17 SEQ ID NO: 6 47.8 68 23 7 hsa-mir-25 SEQ ID NO: 7 50 66 22 8 hsa-mir-31 SEQ ID NO: 8 52.4 64 21 9 hsa-mir-1203 SEQ ID NO: 9 71.4 72 21 10 hsa-mir-1307 SEQ ID NO: 10 72.7 76 22 11 hsa-mir-3141 SEQ ID NO: 11 73.7 66 19 12 hsa-mir-1915 SEQ ID NO: 12 90 76 20 13 hsa-mir-2861 SEQ ID NO: 13 89.5 72 19 14 hsa-mir-3196 SEQ ID NO: 14 88.9 68 18 15 hsa-mir-3665 SEQ ID NO: 15 83.3 66 18

2. Multiple individual miRNA Complementary to cDNA on adapter  Sequence and primer  Preparation of the Second Linked Product with Sequence Linked

Four cDNAs arbitrarily selected from the cDNA complementary to SEQ ID NOS: 1 to 15 were ligated through adapter sequences, and a second linkage product in which the primer sequences were ligated to both ends of the linked product was synthesized. Each of the resulting second link products has the following structure.

P5-A5-cDNA1-A3-A5-cDNA2-A3-A5-cDNA3-A3-A5-cDNA4-A3-P3

In the formulas, A3, A5, P3 and P5 are as described above, and cDNA1, cDNA2, cDNA3 and cDNA4 represent one of the cDNAs complementary to SEQ ID NOS: 1-15.

A first ligated product with four complementary DNAs (cDNA) was synthesized (Bioneer, Korea). Eight kinds of the second link products were prepared, and their cDNAs 1 to 4 were constructed as follows.

turn cDNA composition GC content cDNA1 cDNA2 cDNA3 cDNA4 One hsa-mir-16 hsa-mir-3665 hsa-mir-3196 hsa-mir-1915 58.1 2 hsa-mir-1307 hsa-mir-1203 hsa-mir-25 hsa-mir-1 49.4 3 hsa-mir-17 hsa-mir-25 hsa-mir-1307 hsa-mir-1203 51.8 4 hsa-mir-31 hsa-mir-3141 hsa-mir-25 hsa-mir-16 49.1 5 hsa-mir-2861 hsa-mir-25 hsa-mir-1307 hsa-mir-95 52.1 6 hsa-mir-1307 hsa-mir-1203 hsa-mir-7 hsa-mir-9 48.4 7 hsa-mir-1307 hsa-mir-16 hsa-mir-3196 hsa-mir-31 53.2 8 hsa-mir-1307 hsa-mir-2861 hsa-mir-31 hsa-mir-3196 58.0

3. Amplification of the target nucleic acid

PCR was performed using the first and second linked products prepared in the above 1 and 2 as a template to amplify the target nucleic acid. The oligonucleotide of SEQ ID NO: 16 was used as the forward primer and the oligonucleotide of SEQ ID NO: 18 was used as the reverse primer. Quantitative real-time PCR (qRT-PCR) was performed for PCR, and SYBR GREENI was used for real-time confirmation of amplification products.

qRT-PCR was performed for 40 times at 95 ° C for 10 minutes (95 ° C for 15 seconds / 55 ° C for 1 minute) using a Roche LC480 instrument and the ramping rate was 3.3 ° C / sec. The PCR reaction mixture for amplifying the first linkage product contained 0.5 nM of each template cDNA, 500 nM of each forward and reverse primers, and SYBR GREEN master mix (Universal RT, Exiqon, product # 203450). The SYBR GREEN master mix is a mixture of DNA polymerase, buffer, dNTP, and SYBR GREENI supplied by the manufacturer.

The PCR reaction mixture for amplifying the second ligation product contained 0.5 nM of each template cDNA, 500 nM of each forward and reverse primers, and SYBR GREEN master mix (Universal RT, Exiqon, product # 203450). The SYBR GREEN master mix is a mixture of DNA polymerase, buffer, dNTP, and SYBR GREENI supplied by the manufacturer.

The amplified miRNA was determined by measuring the threshold cycle value of the threshold cycle.

4. Amplification Result

The amplification results of the first link product are shown in Table 3 for the variance (CV) (%) value relative to the mean Ct value.

All 15 species miRNA Mean Ct 21.43 CV (%) 10.92 GC content 20-80% miRNA Mean Ct 20.73 CV (%) 5.83 GC content over 80% miRNA Mean Ct 23.37 CV (%) 16.03

As shown in Table 3, the Ct value varies depending on the GC content of the target nucleic acid. Eleven miRNAs belonging to the 20% to 80% GC content had an average Ct value of 20.73 and a dispersion value of 5.83%, while four miRNAs (hsa-mir-1915 , 2861, 3195 and 3196) had an average Ct value of 23.37 and a variance of 16.03%. This indicates that as the GC content increases, the dispersion value increases. That is, the amplification bias is present according to the sequence of the target nucleic acid, that is, the GC content.

The amplification results of the second link product are shown in Table 4.

turn GC content Ct One 58.1 20.23 2 49.4 18.40 3 51.8 20.67 4 49.1 21.57 5 52.1 19.08 6 48.4 20.56 7 53.2 21.25 8 58 21.81

When the second link product was amplified, the miRNA had an average Ct value of 20.44 and a dispersion value of 5.83%, which was similar to the amplification result of 20-80% GC content of the first link product. This indicates that amplification of the second linkage product can cancel out the amplification bias of the miRNA, respectively, resulting from the difference in the base sequence including the GC content.

Example 2 : miRNA Connection and amplification

Eight kinds of miRNAs with different GC content were selected as the target nucleic acid to be amplified. The 8 selected miRNAs are shown in Table 2.

turn miRNA designation SEQ ID NO: GC content Tm Length One hsa-mir-95 One 36.4 60 22 2 hsa-mir-1307 10 72.7 76 22 3 hsa-mir-1203 9 71.4 71.4 21 4 hsa-mir-4271 19 63.2 62.0 19 5 hsa-mir-1915 12 90 76 20 6 hsa-mir-3141 11 73.7 66 19 7 hsa-mir-2861 13 89.5 72 19 8 hsa-mir-3665 15 83.3 66 18 Average 72.48 Standard Deviation 17.28

The miRNAs in Table 5 were synthesized miRNAs (Bioni, Korea), and the concentrations were about 1 pmole / ul, and were stored at -20 ° C with 5'-phosphorylation and 3'-OH. An adapter sequence and / or a primer sequence was ligated to the miRNA and an amplification reaction was performed. The sequence used for the ligation and amplification is as follows.

P5-A5: 5'-CGGUGAGGUCUUUGGUUCAUCGAUCG-3 '(SEQ ID NO: 20)

A3-P3: 5'-CGAUCGUGUCCUCAAGGCUACCACCU-3 '(SEQ ID NO: 21)

The reverse transcription primer sequence: 5'-ATCGCGAGAATTCCA-3 '(SEQ ID NO: 22)

PCR forward primer: 5'-CGGTGAGGTCTTTGGTTCAT-3 '(SEQ ID NO: 23)

PCR reverse primer: 5'-AGGTGGTAGCCTTGAGGACA-3 '(SEQ ID NO: 24)

P5-A5, A3-P3, reverse transcription and PCR primers were purchased from Bioneer (Korea).

(1) A3- P3  Connection of sequences

The 8 kinds of miRNA shown in Table 5 were mixed at a concentration of 1 pmole / ul, and then 1ul of the mixture and 1ul of the A3-P3 (160 fmole / ul) were mixed. Next, the mixture was added to a reaction buffer (NEB, 1xT4 RNA ligase reaction buffer supplemented with 1 mM ATP) containing 10 units of T4 RNA ligase, and RNA coupling reaction was performed at 37 ° C for 2 hours.

Here, a ratio of A3-P3 to total miRNA mass was used at a rate that 4 miRNAs can be randomly linked in the 5 'direction with A3-P3 connected to the 3' end (1/6 of the total miRNA Using an adapter). MiRNA-miRNA-A3-P3, miRNA-miRNA-A3-P3, miRNA-miRNA-miRNA-A3-P3, , miRNA-miRNA-miRNA-miRNA-miRNA-miRNA-A3-P3, and the like. The 3 'end of -A3-P3 is blocked with 3'-ddC (Dideoxycytidine).

(2) at the 5 'end P5 Linking of -A5 sequence

To the reaction product obtained in (1), 10 pmole / ul of P5-A5 1ul was added and the P5-A5- sequence was ligated to the 5 'end under the same reaction conditions as in (1). As a result, P5-A5-miRNA-miRNA-A3-P3, P5-A5-miRNA-miRNA-miRNA-A3-P3, P5-A5- miRNA- miRNA- miRNA-miRNA-miRNA-A3-P3 and the like (hereinafter referred to as the first linkage product) were generated.

A3-P3 and P5-A5- were linked to the 3 'and 5' ends according to (1) and (2) for each of the 8 kinds of miRNAs as a control group to construct P5-A5-miRNA-A3 -P3. ≪ / RTI >

(3) Reverse transcription  reaction

The reverse transcription reaction was performed using the reverse primer of SEQ. ID. NO. 22 for the first and second products of the test group obtained in (2) and the control product of the control group.

The reverse transcription reaction was carried out for 30 minutes at 16 ° C and 30 minutes at 42 ° C according to the protocol of TaqMan ^™ MicroRNA Reverse Transcription Kit (MultiScribe ^™ Reverse Transcriptase, 50 U / ul, Applied Bioscience; 4366596) Lt; RTI ID = 0.0 > 85 C < / RTI > for 5 minutes. As a result, a cDNA product was obtained.

(4) Amplification and results

Quantitative RT-PCR (qRT-PCR) was performed using the cDNA product obtained in (3) as a template and the sequences of SEQ ID NOS: 23 and 24 as forward and reverse primers (900 nM, respectively). qRT-PCR is ^{Applied Biosystems Taqman TM Universal PCR Master Mix} II, No UNG: was carried out using (part number # 4440043) kit and, Taqman probe 250nM (custome produced from Applied Biosystems). The PCR conditions were denaturation at 95 ° C for 10 minutes, denaturation at 95 ° C for 10 seconds, annealing at 60 ° C for 10 seconds, and polymerization for 45 times. The amplified product was electrophoresed using an Agilent BioAnalyzer 2100.

Fig. 1 is a diagram showing the results of electrophoresis and analysis of amplification products. 1A is an electrophoresis photograph, and FIG. 1B is an electropherogram of 1A. In Figure 1B, a is the experimental group, b is the result of the control group, and 3, 4, and 5 are the result of one miRNA ligation product (about 74 bp), two miRNA ligation products (about 94 bp) (About 154 bp). As shown in Fig. 1B, unlike the control group, the production of by-products was decreased because -A3-P3 was not used excessively in the experimental group.

Thus, as shown in FIG. 2, it was confirmed that in the case of the experimental group, an average of 4 to 5 miRNA-linked first ligated products were obtained. In the case of Example 2, 4 to 5 miRNAs were carried out under conditions that can be linked.

-A3-P3 or P5-A5- is 26 nt in length, and the length of miRNA is 18-22 nt. Therefore, when only one miRNA is linked to -A3-P3 or P5-A5-, , The length of the connection is about 88 to 96 nt when connected, about 106 to 118 nt when three miRNAs are connected, about 124 to 140 nt when four miRNAs are connected, and about 142 to 162 nt when five miRNAs are connected The product is expected to be produced.

Since the linking reaction is a random linkage, the linking product with the length above can be made in various ways, but initially has a size distribution dependent on the amount of -A3-P3. For example, when miRNA and -A3-P3 participate together in the ligation reaction, when the amount of total miRNA and the amount of -A3-P3 have a ratio of 1: 1, the ratio of miRNA and -A3-P3 is 1: 1 , With one -A3-P3 ligated together whenever 5-6 randomly selected miRNAs are ligated by putting the amount of A3-P3 at 1/6 of the total amount of miRNA have. Since -A3-P3 is blocked by dideoxycytidine (3'-ddC) at the 3 'end, it can not be ligated to the 3' end of A3-P3, The reaction is terminated. Therefore, when all of -A3-P3 participates in ligation, ligation does not occur because there is no 3'-OH to lyse with 5'-P of miRNA. However, it is not limited to a specific mechanism.

Table 6 shows the results of qRT-PCR for the experimental group and the control group as ΔCt values.

turn miRNA designation The control (? Ct) The experimental group (Ct) One hsa-mir-95 2.912 0.995 2 hsa-mir-1203 -1.218 0.245 3 hsa-mir-1307 1.812 0.695 4 hsa-mir-1915 -0.198 -0.035 5 hsa-mir-2861 -2.128 -0.765 6 hsa-mir-3141 1.422 -0.205 7 hsa-mir-3665 -1.998 -0.945 8 hsa-mir-4271 -0.608 0.015 Max-min of Ct 5.04 1.94

Ct represents a maximum value-minimum value. The Ct of each experimental group is the value measured in the instrument, and ΔCt is the average Ct of individual miRNAs, calculated as the Ct of individual miRNAs. Therefore, in the control group, the Ct of all the control groups is averaged and then the Ct of each control group is subtracted. The experimental group also shows the deviation from the average of the Ct averages of the respective experimental groups minus the Ct of each experimental group. The max-min of ΔCt is the Ct difference between miRNA with the highest Ct value and the small miRNA in the control and experimental groups. Theoretically, when this value is 0, there is no difference in amplification efficiency between miRNAs. The difference in the amount of amplification is 10 times that of every day.

As shown in Table 6, in the case of the control group, ΔCt is 5.04, even though the same amount of miRNA was used, indicating that the difference in expression amount between individual miRNAs is 32.9 times. In contrast, in the experimental group, ΔCt was 1.94, which was 3.8 times the difference in expression between individual miRNAs. That is, the bias of the experimental group was reduced to 1/10 of that of the control group.

From the above experimental results, it was possible to reduce amplification bias by connecting a plurality of miRNAs after amplification.

<110> Samsung Electronics Co., Ltd. <120> Method for amplifying a target nucleic acid with reduced          amplification bias and method for determining relative amount of          a target nucleic acid in a sample <130> PN093126 <160> 24 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> RNA <213> Homo sapiens <400> 1 uucaacgggu auuuauugag ca 22 <210> 2 <211> 23 <212> RNA <213> Homo sapiens <400> 2 uggaagacua gugauuuugu ugu 23 <210> 3 <211> 22 <212> RNA <213> Homo sapiens <400> 3 uggaauguaa agaaguaugu au 22 <210> 4 <211> 23 <212> RNA <213> Homo sapiens <400> 4 ucuuugguua ucuagcugua uga 23 <210> 5 <211> 22 <212> RNA <213> Homo sapiens <400> 5 uagcagcacg uaaauauugg cg 22 <210> 6 <211> 23 <212> RNA <213> Homo sapiens <400> 6 caaagugcuu acagugcagg uag 23 <210> 7 <211> 22 <212> RNA <213> Homo sapiens <400> 7 cauugcacuu gucucggucu ga 22 <210> 8 <211> 21 <212> RNA <213> Homo sapiens <400> 8 aggcaagaug cuggcauagc u 21 <210> 9 <211> 21 <212> RNA <213> Homo sapiens <400> 9 cccggagcca ggaugcagcu c 21 <210> 10 <211> 22 <212> RNA <213> Homo sapiens <400> 10 acucggcgug gcgucggucg ug 22 <210> 11 <211> 19 <212> RNA <213> Homo sapiens <400> 11 gagggcgggu ggaggagga 19 <210> 12 <211> 20 <212> RNA <213> Homo sapiens <400> 12 ccccagggcg acgcggcggg 20 <210> 13 <211> 19 <212> RNA <213> Homo sapiens <400> 13 ggggccuggc ggugggcgg 19 <210> 14 <211> 18 <212> RNA <213> Homo sapiens <400> 14 cggggcggca ggggccuc 18 <210> 15 <211> 18 <212> RNA <213> Homo sapiens <400> 15 agcaggugcg gggcggcg 18 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> P5-A5: 5 'terminal primer-5' terminal adapter sequence <400> 16 tgagttctac ggtacctcta agc 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> A3-P3: 3 'terminal adaper-3' terminal primer sequence <400> 17 aggcataagc tgttagctca gaa 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer: sequence complemtary to P3-A3 sequence <400> 18 attctgagct aacagcttat gcct 24 <210> 19 <211> 19 <212> RNA <213> Homo sapiens <400> 19 gggggaagaa aaggugggg 19 <210> 20 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> P5-A5: primer or primer binding sequence linked to 5 terminus          Adapter Sequence <400> 20 cggugagguc uuugguucau cgaucg 26 <210> 21 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> A3-P3: 3 terminus adapter sequence linked to a primer or a primer          binding sequence <400> 21 cgaucguguc cucaaggcua ccaccu 26 <210> 22 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> reverse transcription primer <400> 22 atcgcgagaa ttcca 15 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 23 cggtgaggtc tttggttcat 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 24 aggtggtagc cttgaggaca 20

Claims (19)

Providing a biological sample comprising a plurality of types of target nucleic acids, wherein the target nucleic acid is a miRNA;
Linking two or more kinds of the target nucleic acids from the biological sample or cDNAs synthesized therefrom to obtain a resultant product; And
&Lt; / RTI &gt; amplifying the target nucleic acid. delete 2. The method of claim 1, wherein the linking is in the presence of one or more of RNA ligase and DNA ligase. 2. The method of claim 1, wherein the linkage mediates adapter sequences between the target nucleic acid and the target nucleic acid. 2. The method according to claim 1, wherein the linkage comprises a linker between a 3 'end of the target nucleic acid and an adapter sequence specific to the 3' end, and a 5 'end of the target nucleic acid is linked to a target nucleic acid to which an adapter sequence specific to the 5' How to do it. 2. The method of claim 1, wherein a primer sequence or a primer binding sequence is ligated to at least one of the 3 ' and 5 ' ends of the linked product. 7. The method of claim 6, wherein at least one of the primer sequence and the primer binding sequence is specifically linked to either the 3 'end or the 5' end. 2. The method of claim 1, wherein the target nucleic acid is less than 200 nt in length. 2. The method of claim 1, wherein the target nucleic acid is 18 to 30 nt in length. 2. The method of claim 1 wherein the amplification amplifies the linked product as an amplification product. 7. The method of claim 6, wherein the amplification comprises using as primers at least one of the primer sequence and the sequence complementary to the primer binding sequence. The method of claim 1, comprising reverse transcribing a plurality of said target nucleic acid miRNAs to produce complementary DNA (cDNA) to said target nucleic acid. 13. The method of claim 12, wherein the reverse transcription is performed before or after the connecting step. 13. The method of claim 12, further comprising ligating the adapter sequence to one or more of the 3 'and 5' ends of the target nucleic acid prior to reverse transcription. 15. The method of claim 14, wherein the adapter sequence is specifically linked at one or more of the 3 ' and 5 ' ends. 13. The method of claim 12, further comprising ligating the adapter sequence to one or more of the 3 'and 5' ends of the cDNA complementary to the target nucleic acid after the reverse transcription. 17. The method of claim 16, wherein the adapter sequence is specifically linked at one or more of the 3 ' end and the 5 ' end. 2. The method of claim 1 wherein the amplification is PCR and the forward and reverse primers specifically bind to a region other than the target nucleic acid. Amplifying the target nucleic acid in the biological sample according to the method according to any one of claims 1 to 19;
Determining the amount of target nucleic acid from the resulting amplified product; And
Determining the relative amount of the target nucleic acid in the biological sample from the amount of the target nucleic acid.

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