KR20050003502A - Process for improving efficiency of DNA amplification reaction - Google Patents

Abstract

PURPOSE: A process for improving efficiency of DNA amplification reaction is provided, thereby simplifying a procedure for deciding optimal temperature(annealing) condition and improving hybridization specificity of oligonucleotides to DNA. CONSTITUTION: The process for improving efficiency of a DNA amplification reaction comprises using a primer in which a compound is added into the 5'-terminal of the primer, wherein the compound is selected from LC-Red 705, amino group, phosphate group, biotin, DIG, DNP, TAMRA, Texas-Red, ROX, XRITC, rhodamine, LC-Red 640, mercapto group, psoralen, cholesterol, FITC, 6-FAM, TET, cy3, cy5, BODIPY 564/570, BODIPY 500/510, BODIPY 530/550, BODIPY 581/591 and oligonucleotides having a combined G and C content of at least 25% and having at least four bases.

Description

Process for improving efficiency of DNA amplification reaction

The present invention relates to a method for improving the efficiency of a DNA amplification reaction, and to a method for improving the hybridization specificity of oligonucleotides for a DNA sample.

DNA amplification is extremely important in the detection of genes, and PCR methods in the field of DNA amplification enable large-scale amplification of target sites of nucleotide sequences in DNA, and are used in a variety of other fields as well as in life science technology.

However, if specific detection primers are designed, they should contain base positions specific for the target sequence.

Unfortunately, sequences comprising this type of position are often unsuitable as primers. In other words, for example, when the AT content is extremely high or when the front and reverse melting temperatures (Tm) do not match, the amplification efficiency is lowered and the sequence cannot be used as a primer. This problem is a significant disadvantage when it is necessary to detect very small amounts of DNA.

Moreover, in the PCR method, the optimum temperature condition for amplification must be determined in order to improve the amplification efficiency. However, determining the optimal temperature conditions requires a complex series of preliminary tests.

Accordingly, it is an object of the present invention to provide a method for simplifying the operation for determining the optimum temperature condition and improving the efficiency of the DNA amplification reaction.

It is also an object of the present invention to provide a method for improving the hybridization specificity of oligonucleotides to DNA.

1 is a graph showing the relationship between the number of PCR cycles and fluorescence intensity under annealing conditions at 60 ° C. for 0 seconds.

2 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity at annealing conditions of 60 ° C. for 5 seconds.

3 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity at annealing conditions of 60 ° C. for 10 seconds.

4 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity at annealing conditions of 64 ° C. and 5 seconds.

The inventors of the present invention have found very surprising facts. That is, when the artificial nonspecific sequence is added to the 5 'end of the degenerate primer, the PCR amplification efficiency increases, and when the sequence added to the 5' end is removed, the PCR amplification efficiency decreases.

As a result, the present inventors have conducted further studies to improve PCR amplification efficiency by not only adding artificial non-specific sequences but also conjugating a compound such as LC-Red 705 to the 5 'end, thereby amplifying the DNA amplification reaction. It was found by chance to improve the efficiency of the present invention was completed accordingly. The optimum temperature range for annealing can be widened, which means that the preliminary tests for investigating the annealing conditions can be simplified and the overall process can be significantly simplified.

In other words, the first aspect of the present invention provides a method for improving the efficiency of the DNA amplification reaction, wherein the primer is LC-Red 705, amino group, phosphate group, biotin, DIG, DNP, TAMRA, Texas-Red, ROX, XRITC, Rhodamine, LC-Red 640, mercapto group, psoralen, cholesterol, FITC, 6-FAM, TET, cy3, cy5, BODIPY 564/570, BODIPY 500/510, BODIPY 530/550, BODIPY 581/591 (" A compound selected from the group consisting of oligonucleotides having a GC content of at least 25% and having at least four bases (hereinafter referred to as "specificated bases") as primers. use.

A second aspect of the present invention provides a method for improving the hybridization specificity of oligonucleotides for a DNA sample, wherein oligonucleotides added at the 5 ′ end of a compound selected from the specified group of compounds are used for hybridization with DNA.

In the first and second aspects of the present invention, if it corresponds to a primer or oligonucleotide that is typically used in DNA amplification, it is specific to primers or oligonucleotides to which a compound selected from a group of specified compounds and a specified base are conjugated or added. There is no limit.

Furthermore, since the present invention is intended to improve the DNA amplification efficiency, some primers which are not normally available for DNA amplification can also be used.

In the first and second aspects of the invention, the compounds of the specified group of compounds are nonspecific for the target sequence to be amplified by DNA amplification. DIG stands for digoxigenin, DNP stands for dinitrophenyl, TAMRA stands for carboxytetramethylhodamine, Texas-Red stands for 1H, 5H, 11H, 15H-xanteno [2,3, 4-ij: 5,6,7-i'j '] diquinolizine-18-ium, 9- [2 (or 4)-[[[6- (2,5-dioxo-1-pyrrolidinyl ) Oxy] -6-oxohexyl] amino] sulfophenyl] -4 (or 2) sulfonyl] -2,3,6,7,12,13,16,17-octahydro-, inner salt , ROX stands for Rhodamine X, XRITC stands for Rhodamine X isothiocyanate, FITC stands for fluorescein isothiocyanate, 6-FAM stands for 6-carboxyfluorescein, TET Abbreviation for tetrachlorofluorescein, BODIPY 564/570 is 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, succinimidyl ester BODIPY 530/550 is 4,4-difluoro-5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, succinimidyl ester, BOD IPY 581/591 is 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, succinate Imidyl ester.

According to the first aspect of the invention, the specified base may be specific or nonspecific to the nucleotide sequence to be hybridized. Here, "specific" means that the additional sequence for the primer hybridizes as well as the addition sequence for the primer is complementary to the template region that is not adjacent to the region where the primer hybridizes. This includes complementary to template areas adjacent to the area (particularly the 3 'area of the template corresponding to the 5' area of the primer). The latter case is used in the prior art to adjust the Tm value of the primer, but in the present invention is used to improve the amplification efficiency.

Nonspecific includes when there is no relationship between the primer and the template, ie, when the additional sequence has a nucleotide sequence in which a double strand is not formed adjacent to the GC and AT base pairs. Even in this non-specific case, the present invention can increase the highest annealing temperature of the primer. Thus, in the first aspect of the invention, the specified base may be specific or nonspecific to the nucleotide sequence to be hybridized (template DNA). However, in order to increase the amplification efficiency, the specified sequence is preferably nonspecific.

In a first aspect of the invention, one or more compounds selected from the group of specified bases and specified compounds can be used.

In a second aspect of the invention, one or more compounds selected from the group of specified compounds can be used.

In the first and second aspects of the invention, compounds selected from the group of oligonucleotides or primers and specified compounds may be conjugated via a linker. Examples of suitable linkers include hydrocarbon groups of 2 to 16 carbon atoms.

In the first aspect of the invention, the oligonucleotide (additional sequence) added to the 5 'end of the primer preferably has a high GC content. Specifically, the oligonucleotides should have a GC content of at least 50%, with addition sequences of at least four bases also being preferred. The efficiency of the DNA amplification reaction is improved as the GC content increases, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. Is even more preferred. Anyone skilled in the art can determine the GC content based on the length of the additional sequence and the non-complementary between the additional sequence and the sequence or primer to be amplified. If the additional sequence is too long, the efficiency of the DNA amplification reaction decreases, and an additional sequence of 40 bases or less is typically preferred. Moreover, in order to prevent the formation of primer dimers, the amount of G or C is preferably at least 50% and the amount of A or T is preferably at least 50%.

Specific examples of preferred nucleotide sequences are described below. Moreover, sequences of 9 bases or more can be made by appropriate combination of the following 2 to 8 nucleotide sequences. In the following sequences, S represents C or G, and W represents A or T. The amount of G or C preferably accounts for at least 50% and the amount of A or T preferably accounts for at least 50%.

SS, SW, WS, SSS, SSW, SWS, WSS, SSSS, SSSW, SSWS, SWSS, WSSS, SSSSS, SSSSW, SSSWS, SSWSS, SWSSS, WSSSS, SSSSSS, SSSSSW, SSSSWS, SSSWSS, SSWSSS, SWSSSS, WSSSSS, SSSSSSS, SSSSSSW, SSSSSWS, SSSSWSS, SSSWSSS, SSWSSSS, SWSSSSS, WSSSSSS, SSSSSSSS, SSSSSSSW, SSSSSSWS, SSSSSWSS, SSSSWSSS, SSSWSSSS, SSWSSSSS, SWSSSSSS, WSSSSSSS, SSSSSS, SWSS, SWSS, SSSS SSSSWSWS, SSSWSSWS, SSWSSSWS, SWSSSSWS, WSSSSSWS, SSSSWWSS, SSSWSWSS, SSWSSWSS, SWSSSWSS, WSSSWSS, SSSWWSSS, SSWSWSSS, SWSSWSSS, WSSSWSSS, SSWWSSSS, SWSWSSSS, WSSWSSSS, SWWSSSSS, WSWSSS

Moreover, embodiments of oligonucleotides include up to 20 bases formed from repeat units of AGTC, AAGT, GGAC or GGGC.

Furthermore, it is preferable that the additional sequence has a nucleotide sequence which forms a secondary structure and does not interfere with the amplification reaction. Specifically, it is preferable that the additional sequence shows a low base pair formation rate with respect to the 3 'terminal sequence, and it is more preferable not to form a base pair. Preferred minimal conditions are the absence of continuous base pair formation. It is also preferred that primers with additional sequences exhibit low base pair formation, even more preferably no base pair formation.

In a first aspect of the invention, a compound selected from the group of specified compounds or a specified base can be conjugated or added to the 5 'end of the primer according to standard methods. In order to conjugate or add two or more of the compounds selected from the group of specified compounds or specified bases, alternatively first conjugate the compounds and then synthesize oligonucleotides comprising the specified bases, or first specify the specified bases. Oligonucleotides can be synthesized and subsequently conjugated to compounds. An example of a primer having two or more of a compound selected from the group of specified compounds or an additional specified base is FITC to which a double repeat sequence of GGGC is added.

In a second aspect of the invention, a compound selected from the group of specified compounds can be conjugated to the 5 'end of the oligonucleotide according to standard methods. In order to conjugate two or more of the compounds selected from the group of specified compounds or specified bases, alternatively first conjugate the compound and subsequently synthesize oligonucleotides comprising the specified bases, or first comprise the specified bases. Oligonucleotides can be synthesized followed by conjugation of the compounds.

In a first aspect of the invention, by conjugating a compound selected from a group of specified compounds to a primer or adding a specified base to the primer, the annealing rate and annealing stability of the primer for the amplified product can be improved, which is the primer Means that it is ideally suited for normal PCR. It can be seen that the annealing rate and annealing stability are improved even when the specified bases are not complementary to the template DNA (see results in Table 2).

In addition, the present invention is also ideally applied to asymmetric PCR. Asymmetric PCR is a method used to rapidly amplify single stranded DNA, such as when it is necessary to directly sequence a target DNA fragment. In other words, the concentration of primer pairs used in normal PCR is the same, but in asymmetric PCR, the concentration of one of the primers is several times or tens of times higher than that of the other primers. By doing so, the lower concentration of the primer is consumed first and the remaining PCR proceeds with only the higher concentration of the remaining primers, resulting in a higher amount of DNA strand corresponding to the higher concentration of the primer. Moreover, a specific type of asymmetric PCR, thermal asymmetric PCR, uses a pair of primers that show a difference of at least 10 ° C. in Tm, so that the first PCR is performed under conditions where primers of lower Tm values are annealed, and Subsequent PCR is performed under conditions in which only high Tm primers are annealed.

However, asymmetric PCR has the following types of problems. That is, if the concentration of template DNA and primers is not optimized, the amplification of single strands is low (Production of Single Stranded DNA by Asymmetric PCR, PCR Protocols, A guide to Methods and Applications, Academic Press, Inc. 1990). However, such optimization requires complex preliminary testing.

In addition, in thermal asymmetric PCR, a set of specific primers having a large difference in annealing temperature of at least 10 ° C. must be prepared, which is not necessarily a simple task.

Another method of rapidly amplifying single stranded DNA is to exploit the difference in amplification capacity within primer pairs. For example, hybrid primers of DNA and RNA can be used. RNA primers contribute less to extension reactions than DNA primers, so PCR amplification with this type of hybrid primer results in greater amplification toward pure DNA resulting in single stranded DNA.

However, this method results in single stranded DNA due to the low amplification capacity on the RNA side and is not due to improved amplification capacity of the desired DNA.

On the other hand, in the first aspect of the present invention, by conjugating a compound selected from a group of compounds specified to only one of the primer pairs or adding a specified base, a large difference in amplification efficiency can be generated between the two primers, which means that the template DNA And complex manipulations to optimize primer concentrations. As a result, by applying the first aspect of the present invention to asymmetric PCR, the amplification efficiency for single stranded DNA can be greatly improved. Moreover, the first aspect of the present invention allows the optimum temperature range for primers to be broadened, allowing the present invention to be applied to thermal asymmetric PCR.

Conventionally, asymmetric PCR has been performed by inhibiting elongation of one of the primers, ie effectively lowering overall PCR efficiency. On the other hand, the first aspect of the present invention allows to perform asymmetric PCR by improving the amplification efficiency of one of the primers. In other words, compared with the conventional PCR, the asymmetric PCR using the present invention does not suffer a decrease in amplification efficiency. Therefore, the first aspect of the present invention is required when a single strand of DNA needs to be produced while maintaining a high level of amplification efficiency, such as when a trace amount of DNA is rapidly and easily detected and its type needs to be determined, such as a pathogen. Is particularly effective.

In addition, the present invention can also be ideally applied to analogy PCR (deganerate PCR). In analogy PCR, hundreds to thousands of different primer mixtures are used, meaning that the optimum annealing temperature will be different for each sequence in the mixture. As a result, the setting of the amplification temperature is relatively difficult. However, this type of problem can be solved using the present invention. Moreover, since the annealing temperature can be set at a relatively high temperature, nonspecific amplification can be suppressed and more effective amplification can be made.

Primers conjugated with compounds selected from the group of specified compounds of the first aspect of the invention may also be used as primers.

In a second aspect of the invention, by using oligonucleotides conjugated at the 5 ′ end of a compound selected from the group of specified compounds, the hybridization specificity of oligonucleotides to DNA can be improved. In other words, compared to oligonucleotides to which the specified compounds are not conjugated, oligonucleotides to which the specified compounds are conjugated improve both hybridization rate, and hybridization stability to DNA.

Example

The present invention is explained in more detail based on a series of examples. However, the present invention is not limited to the following examples.

Example 1

(Comparison of Upper Annealing Temperatures for Amplification)

PCR Express instrument (Hybaid Co., Ltd) using a gradient block module added using a primer for detecting hemolytic Vibrio parahaemolyticus, to which a compound selected from the compound group specified at the 5 'end is conjugated. Using the above, the upper annealing temperature of the amplification was measured by performing PCR under the following conditions.

The hemolytic Vibriovirus detection primers used the nucleotide sequence shown in SEQ ID NO: 1 as the forward primer and the nucleotide sequence shown in SEQ ID NO: 2 as the reverse primer.

SEQ ID NO: 1: aagaagacct agaagatgat

SEQ ID NO: 2 gttaccagta atagggca

Each compound of the specified group of compounds shown in Table 1 was conjugated to the 5 'end of the forward and reverse primers. In a separate preparation, chromosomal DNA extracted from one type of strain of hemolytic Vibrio strain (IFO12711T) was used as a template, and rpoD gene amplification universal primer (Japanese Patent Application Laid-open No. Hei 8-256798: SEQ ID NO: 3 and PCR was performed using 4) to prepare amplified products. The PCR test was then performed under a plurality of different annealing temperature conditions using the amplification product as a template and the primer to which the compound from the specified compound group was added.

SEQ ID NO: 3: yatgmgngar atgggnacngt

(y represents base T or U, or C; m represents A or C; n represents A, C, G, or T or U)

SEQ ID NO 4: ngcytcnaccatytcyttytt

PCR conditions are as follows. (1) activation of Taq polymerase (AmpliTaq Gold, Applied Biosystems Co., Ltd.): 10 min at 95 ° C. (2) Double helix separation (denaturation): 1 minute at 94 ℃. (3) Annealing: 30 seconds at 55.1 ° C, 55.5 ° C, 56.3 ° C, 57.7 ° C, 59.4 ° C, 61.4 ° C, 63.3 ° C, 65.3 ° C, 67.6 ° C, 69.0 ° C, 69.7 ° C and 70.2 ° C. 1 min at 72 ° C. Steps (2) to (4) were repeated 40 cycles. (5) Extension reaction: 10 minutes at 72 degreeC. (6) Cooling: Cool to 4 ° C.

Then, the amplified fragment thus obtained was analyzed by agarose gel electrophoresis, and the upper limit annealing temperature was determined. Primers to which the compound derived from the specified compound were not conjugated were used as a control. The results for the upper annealing temperature and the temperature increase of the upper annealing temperature relative to the control value are shown in Table 1.

Conjugated compound Linker effect Upper limit annealing temperature Temperature increase (compared to control primer) BODIPY564 / 570 AA (max) 63.3 ℃ 5.6 ℃ LC-Red705 none A 63.3 ℃ 5.6 ℃ Amino group C3 C3 A 61.4 ℃ 3.7 ℃ Phosphate none A 61.4 ℃ 3.7 ℃ Biotin C10 A 61.4 ℃ 3.7 ℃ DIG C6 A 61.4 ℃ 3.7 ℃ DNP C14 A 61.4 ℃ 3.7 ℃ TAMRA C6 A 61.4 ℃ 3.7 ℃ Texas-red C6 A 61.4 ℃ 3.7 ℃ ROX C6 A 61.4 ℃ 3.7 ℃ XRITC (Rhodamine 600) C6 A 61.4 ℃ 3.7 ℃ Rhodamine C6 A 61.4 ℃ 3.7 ℃ LC-Red640 C6 A 61.4 ℃ 3.7 ℃ BODIPY500 / 510 A 61.4 ℃ 3.7 ℃ BODIPY530 / 550 A 61.4 ℃ 3.7 ℃ BODIPY581 / 591 A 61.4 ℃ 3.7 ℃ Amino group C6 C6 B 59.4 ℃ 1.7 ℃ SH (towel) none B 59.4 ℃ 1.7 ℃ Psoralen C2 C2 B 59.4 ℃ 1.7 ℃ Psoralen C6 C6 B 59.4 ℃ 1.7 ℃ cholesterol B 59.4 ℃ 1.7 ℃ FITC C6 B 59.4 ℃ 1.7 ℃ 6-FAM C6 B 59.4 ℃ 1.7 ℃ TET C6 B 59.4 ℃ 1.7 ℃ cy3 C3 B 59.4 ℃ 1.7 ℃ cy5 C3 B 59.4 ℃ 1.7 ℃ No label (control) - 57.7 ℃ -

AA: very good

A: Excellent

B: good

Primers to which the compounds derived from the specified compound group were added showed that the upper annealing temperature was increased and the amplification efficiency was improved.

Example 2

(Comparison of Upper Annealing Temperatures for Amplification)

The nucleotide sequence shown in SEQ ID NO: 3 was used as the forward primer and the nucleotide sequence shown in SEQ ID NO: 4 was used as the reverse primer. PCR Express apparatus (Hybaid Co., Ltd.) to which 4-mers to 20-mers having the nucleotide sequence shown in Table 2 as repeating units were added to the 5 'end of each primer and a gradient block module was added. Using the chromosomal DNA extracted from one type of strain of hemolytic Vibrio (IFO12711T) as a template and subjected to PCR under the same conditions as in Example 1 with annealing time of 1 minute to determine the upper annealing temperature for amplification. Primers without added oligonucleotides were used as controls. In addition, primers containing 4-mers to 20-mers of the repeating unit AAAT having a GC content of 0% were used for comparison purposes. The results for the upper annealing temperature and the temperature increase (in parentheses) of the upper annealing temperature for the control values are shown in Table 2.

Repeat sequence AGTC AAGT GGAC GGGC AAAT none x1 (4-mer) 63.3 (+1.9) 63.3 (+1.9) 65.3 (+3.9) 67.6 (+6.2) 61.4 (± 0) x2 (8-mer) 63.3 (+1.9) 63.3 (+1.9) 65.3 (+3.9) 69 (+7.6) 59.4 (-2.0) x3 (12-mer) 65.3 (+3.9) 63.3 (+1.9) 67.6 (+6.2) 63.3 (+1.9) 59.4 (-2.0) x4 (16-mer) 63.3 (+1.9) 63.3 (+1.9) 63.3 (+1.9) N.A. 57.7 (-3.7) x5 (20-mer) 65.3 (+3.9) 63.3 (+1.9) 57.7 (-3.7) N.A. 57.7 (-3.7) GC% 50% 25% 75% 100% 0% 61.4

N.A. Not Amplified

Primers comprising additional oligonucleotides having at least 25% GC content and at least 4 bases showed improved amplification efficiency, and this effect was particularly pronounced for primers containing additional oligonucleotides having high GC content.

Example 3

(Investigation of Amplification Efficiency in Primer Containing Compound Selected from Specified Compound Group Added or Specified Base at 5 'End)

The nucleotide sequence shown in SEQ ID NO: 1 was used as the forward primer and the nucleotide sequence shown in SEQ ID NO: 2 was used as the reverse primer. 12-mer, G3, cy3, cy5, biotin, or 12-mer with AAGT in the repeat unit is added (conjugated) to the 5 'end of the forward and reverse primers and the Light Cycler System, Roche Diagnostics Co., Ltd.) and chromosomal DNA extracted from one type of hemolytic Vibrio strain (IFO12711T) as a template to perform real-time PCR under the following conditions to analyze the kinetics of the amplification reaction It was.

PCR conditions were as follows. (1) Double helix separation (denaturation): 1.5 minutes at 95 ℃. (2) Double helix separation (denaturation): 0 minutes at 95 ℃. (3) Annealing: 0, 5, or 10 seconds at 60 ° C, or 5 seconds at 64 ° C. (4) Elongation reaction: 15 seconds at 72 ° C.

Steps (2) to (4) were repeated 40 cycles.

The fluorescence intensity was measured for the amplified fragments obtained after each cycle. Primers without additional compounds from the specified bases were used as controls. The results are shown in FIGS. 1 to 4. This fluorescence intensity is not derived from compounds of the specified group of compounds conjugated to primers but from intercalated cyber greens and represents the amount of DNA amplified.

1 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity under annealing conditions at 60 ° C. for 0 seconds.

Fig. 2 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity under annealing conditions at 60 ° C. for 5 seconds.

3 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity under annealing conditions at 60 ° C. for 10 seconds.

4 is a graph showing the relationship between the number of PCR cycles and the fluorescence intensity under annealing conditions of 64 ° C. and 5 seconds.

Under all amplification conditions, primers (modified primers) with added or specified bases selected from the group of specified compounds are primers (unmodified primers) without addition of the selected compounds from the specified compound group or without specified bases. It shows a faster rise in amplification reaction than). In other words, when a modified primer is used, the number of cycles required to exhibit a constant fluorescence is shortened (see Figs. 2 and 3).

If the annealing time is shortened for the same annealing temperature (from 10 seconds (FIG. 3) to 5 seconds (FIG. 2) to 0 seconds (FIG. 1)), the cycle in which amplification of the unmodified primers is started is significantly delayed (in turn) In other words, amplification is attenuated). For example, to achieve fluorescence intensity above 10, 14 cycles are required for annealing time of 10 seconds (FIG. 3), while 20 cycles are needed for annealing time of 5 seconds (FIG. 2) and annealing 0 seconds. In the case of time, even after 40 cycles, the fluorescence intensity does not exceed 10. In contrast, in the case of modified primers, the delay of the amplification initiation cycle due to the shortening of the annealing time is significantly less than that seen with unmodified primers. In other words, when used in a detection system, modified primers provide an increase in sensitivity. This modification effect is particularly pronounced in the case of annealing temperature of 0 seconds, and among the various modification primers, the effect is particularly high for primers to which 12-mers having GCAC are added as repeat units.

When using modified primers, using unmodified primers can achieve substantial levels of amplification even with annealing temperatures and annealing times where no amplification occurs (see FIG. 4). This effect is especially pronounced for primers added with 12-mers having GGAC in repeat units.

The final amount of amplification product after 40 cycles is significantly higher for modified primers than for unmodified primers (see Figures 1-4). In conventional PCR, amplification reactions are usually not performed to more than 40 cycles, so modified primers provide a clear advantage within a substantial cycle range.

The results show the effect of the modification under conditions of both increased temperature and shortened annealing time, which is believed to be due to the improvement of the thermal stability of the hybridization between the primer and the template DNA, ie the increase in the bond strength.

According to the first aspect of the present invention, the preliminary test for examining the annealing conditions and the like can be considerably simplified, and the PCR amplification efficiency can be improved. This effect is particularly pronounced when the PCR is asymmetric PCR or analogy PCR.

In addition, according to the second aspect of the present invention, the hybridization specificity of oligonucleotides for DNA samples can be improved.

Claims (5)

LC-Red 705, amino group, phosphate group, biotin, DIG, DNP, TAMRA, Texas-Red, ROX, XRITC, rhodamine, LC-Red 640, mercapto group, psoralen, cholesterol, FITC, 6-FAM, TET, cy3 , cy5, BODIPY 564/570, BODIPY 500/510, BODIPY 530/550, BODIPY 581/591 and compounds selected from the group consisting of oligonucleotides having at least 25% combined G and C content and having at least four bases. Method for improving the efficiency of DNA amplification reaction using a primer added to the 5 'end as a primer. The oligonucleotide of claim 1, wherein said oligonucleotide having a combined G and C content of at least 25% and having at least four bases has a combined G and C content of at least 50% and comprises 40 bases or less, wherein The amount of base present in a higher number accounts for at least 50% of the combined G and C content, and the amount of base present in a higher number of A and T comprises at least 50% of the combined A and T content. Characterized in that the efficiency improvement of the DNA amplification reaction. The method for improving the efficiency of a DNA amplification reaction according to claim 1 or 2, which is a method for improving the efficiency of PCR amplification. 4. The method of claim 3, wherein said PCR is one of asymmetric PCR and analogy PCR. LC-Red 705, amino group, phosphate group, biotin, DIG, DNP, TAMRA, Texas-Red, ROX, XRITC, rhodamine, LC-Red 640, mercapto group, psoralen, cholesterol, FITC, 6-FAM, TET, cy3 to the DNA, wherein an oligonucleotide having a compound selected from the group consisting of cy5, BODIPY 564/570, BODIPY 500/510, BODIPY 530/550 and BODIPY 581/591 added at the 5 ′ end is used for hybridization to DNA. Method for improving the hybridization specificity of oligonucleotides for.