KR101772866B1 - Mutant Detection Method by Amplification Signal Enhanced Real-Time Polymerase Chain Reaction - Google Patents
Mutant Detection Method by Amplification Signal Enhanced Real-Time Polymerase Chain Reaction Download PDFInfo
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Abstract
Description
The present invention provides real-time PCR using a NSTS (Novel Single Nucleotide polymorphism (SNP) Typing System) probe (hereinafter referred to as a base-substituted NSTS probe) containing a base that is not complementary to the nucleotide sequence of the template , ≪ / RTI > RT-PCR). More specifically, substituting at least one of the bases constituting the NSTS probe with a template and non-complementary base enhances the efficiency of the PCR amplification and produces a NSTS probe that can realize a more enhanced amplification signal, Substituted NSTS probe with a TagMan probe to further amplify the amplified signal.
NSTS (Novel SNP Typing System) is based on RT-PCR and is a new SNP detection system using FEN (Flap endonuclease) specificity of DNA polymerase. Conventional methods using RT-PCR and hybridization probes or hydrolysis probes (such as hydrolysis probes or TaqMan probes) use the difference in complementary binding force of probes with temperature only to distinguish SNPs, while NSTS It is advantageous in the specificity of the test and in the multi-species analysis by using the characteristics of the enzyme rather than the classification based on the classification (Patent Publication 10-2015-0082848).
RT-PCR using the NSTS probe is one of the methods applicable to the detection of tumor-specific mutations requiring high specificity and sensitivity as a means of detecting gene mutation which is rapid, sensitive and specific and is economical.
(1) the sensitivity to detect mutant DNA present in a low ratio in normal DNA; and (2) the false positive to misjudge normal DNA mutation DNA. false positive rate is minimized as much as possible.
Several methods have been developed and used to detect tumor-specific mutations. Direct sequencing, allele-specific PCR (AS-PCR), restriction fragment length polymorphism Restriction Fragment Length Polymorphism (RFLP), TaqMan probe method, and amplification refractory mutation system (ARMS) PCR. However, these methods failed to show satisfactory results in sensitivity and specificity. Direct sequencing has the disadvantage of being detectable only in the presence of 20 to 30% or more of mutant DNA, while the ratio of false positives is low due to its high specificity. On the other hand, AS-PCR, RFLP, and TaqMan probe methods have high sensitivity but low specificity and always have problems of false positives.
Recently, a peptide nucleic acid-mediated PCR clamping method (Sun, X., et al., 2002. Nat Biotechnol, 20: 186-189), LNA (locked nucleic acid) mediated PCR clamping method (Dominguez, PL, et (Li, J., et al., 2008. Nat Med, 14: 579-584), and the like, as well as COLD-PCR (co-amplification at lower denaturation temperature PCR) However, PNA or LNA-mediated PCR clamping has been limited in its wide application due to limited availability and high cost. COLD-PCR is simple to perform, but the amplification rate is 3-100 times It is unsatisfactory and has a disadvantage that it is weak against a minute change of temperature.
The ARMS-PCR method was developed to improve specificity, which is a disadvantage of AS-PCR. It is based on the principle that PCR is performed only when the primer is composed of a perfect complementary sequence with the sequence of the template as in AS-PCR (Newton, CR, et al., 1989. Nucl Acids Res, 17: 2503-2516). More specifically, when the 3 'terminal base of a primer corresponding to a specific SNP position coincides with a nucleotide sequence of a template The method is based on the principle that the amplification is performed and the amplification is not performed when it does not match. However, it is very difficult to control the PCR amplification by virtue of the coincidence or inconsistency with the template of the base of one base at the 3 'end of the primer. To overcome this problem, a method has been developed which uses a primer with base substitution added to dislocate the third or fourth base from the 3 'end of the primer with the template (Kwok, S., et al. 1990. Nucl Acid Res 18: 999-1005). Typical ARMS primer design guidelines should be designed so that if the 3 'end is a weak mismatch such as G-G, then the added base substitution will be a strong incompatibility such as G-A. Conversely, if the 3 'end is strongly discordant, then the base substitution added should be designed to be a mismatch. According to Little, GA, CT and TT are the strongest discrepancies, CC is the strongest discrepancy, AA, GG is the medium discrepancy, CA, GT is the weak discrepancy, and AT and GC are the weakest discrepancies , 1994. John Wiley & Sons, Inc. New York, pp.9.8.1.-9.8.12.). However, even if the above-mentioned criteria are applied, it is a reality that experimental trial and error is required to determine an optimal primer having excellent specificity (Drenkard, E., et al., 2000. Plant Physiol. 124: 1483-1492). Recently, guidelines for reflecting statistical significance have been developed using 2071 pairs of primers to solve this problem (Liu, J., et al., 2012. Plant Methods. 8: 34).
Although a variety of molecular diagnostic techniques have been developed as described above, there is still a need to develop molecular diagnostic techniques that have sufficient sensitivity and specificity.
In the present invention, an allele-specific probe based on RT-PCR is used, but a method of enhancing the amplification signal to enhance the sensitivity of NSTS, which has advantages in assay specificity and multi-species analysis, by using the specificity of the enzyme, I want to.
For this purpose, the inventors of the present invention confirmed that a base-substituted NSTS probe in which at least one of the bases constituting the NSTS probe was replaced with a template and a non-complementary base enhances RT-PCR amplification efficiency and further amplifies the amplification signal. .
The present invention is intended to improve the sensitivity of NSTS designed to effectively detect mutant DNA such as SNPs.
The present invention provides a method of designing a probe suitable for NSTS based on RT-PCR which can increase the sensitivity.
The present invention is characterized by substituting a base at a specific position of an NSTS probe.
The present invention is characterized by enriching the RT-PCR amplified signal using a base substituted NSTS probe.
The term " base-substituted NSTS probe " in the present invention is collectively referred to as an NSTS probe in which at least one base among the nucleotide sequences constituting the probe is substituted with a template and non-complementary. Specifically, a base in which one of the 7th, 8th, 9th and 10th bases is additionally substituted with a base which is not complementary to the base of the template together with the third base substitution (basic NSTS probe) from the 5'end of the probe Quot; oligonucleotide "
The present invention is characterized by enhancing the RT-PCR amplified signal by using the same fluorescent substance-labeled NSTS probe and TaqMan probe.
The present invention relates to a method for enhancing an amplified signal using a non-fluorescent NSTS probe using SYBR Green.
In addition, the NSTS probe of the present invention is characterized in that the third base from the 5 'end is an oligonucleotide of the (Mis + 3 (2) / Mis3 + (1)) form substituted with a base that is not complementary to the base of the template.
Also, the present invention is characterized in that the NSTS probe is an oligonucleotide in which one of the 7th, 8th, 9th and 10th bases from the 5 'end is substituted with a base that is not complementary to the base of the template.
The present invention relates to a method for enhancing an amplified signal using the NSTS probe in which fluorescent substances FAM and BHQ1 are bound at both ends, respectively.
In addition, the present invention is characterized in that the NSTS probe to which the fluorescent substance is coupled is an oligonucleotide in which the ninth base is substituted from the 5 'end.
In addition, the present invention provides a method for enhancing an amplified signal by simultaneously using a TaqMan probe labeled with the same fluorescent substance as the NSTS probe.
In addition, the present invention provides a method for inspecting SNP by RT-PCR using the above-mentioned NSTS probe.
According to the present invention, a means for further enhancing the sensitivity of NSTS based on RT-PCR is provided.
According to the method of the present invention, the use of a base-substituted NSTS probe increases the PCR efficiency and enhances the RT-PCR amplification signal.
Also, unlike PNAs that can not be labeled with fluorescent materials, NSTS probes can be produced in the form of a fluorescent substance labeled. Therefore, the RT-PCR amplification signal can be enhanced by using the NSTS probe labeled with the same fluorescent substance simultaneously with the TaqMan probe, which is a means for further enhancing the sensitivity of NSTS.
The sensitivity-enhanced NSTS of the present invention can be used as an assay for detecting tumor-specific mutations requiring high sensitivity and specificity.
FIG. 1 shows the RT-PCR amplification pattern according to the base substitution position of the NSTS probe.
Fig. 2 shows the RT-PCR amplification pattern according to the type of the 9th substituted base from the 5 'end of the NSTS probe.
FIG. 3 shows the RT-PCR amplification pattern according to the type of the 8th base substituted from the 5 'end of the NSTS probe.
FIG. 4 shows RT-PCR amplification patterns according to the 10th base substitution type from the 5 'end of the NSTS probe.
Fig. 5 schematically shows the types and positions of primers and probes.
FIG. 6 shows the nucleotide substitution of the NSTS probe subjected to the FRET (Fluorescence Resonance Energy Transfer) principle and the RT-PCR amplification pattern according to the addition of the TaqMan probe.
Hereinafter, the configuration of the present invention will be described in more detail with reference to examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.
[Plasmid DNA sample preparation]
A 375 bp PCR product containing the L858R region in the exon 21 region of the human epidermal growth factor receptor (EGFR) gene was prepared and cloned into the pTop TA vector (Enjinomics, Korea) (L858) and mutant (R858) plasmid DNA were prepared, respectively. The EGFR L858R normal and mutant plasmid DNAs were diluted with sterile distilled water to prepare 1x10 4 copies / μl each. The DNA thus prepared was stored frozen until used for experiments.
[ primer And Probe making]
A primer was designed to PCR amplify a specific region of EGFR L858R plasmid DNA. The NSTS probe was designed to have an amplification product and one or two bases that are not complementary sequences but have a base sequence capable of hybridization. We designed a NSTS probe and a TaqMan probe that combine a reporter and a quencher for hydrolysis probe analysis using the Fluorescence Resonance Energy Transfer (FRET) principle. The designed primers and probes were prepared using oligonucleotide synthesis system of Genotech Co., Ltd., which is the present applicant.
[RT- PCR Reaction and confirmation]
Using the prepared EGFR L858R plasmid DNA as a template, real-time PCR was performed using the prepared primers and probes. Primers and probes used were described in each example. The polymerase and the reaction composition used in this example were mixed with 5 x qPCRMix (50 mM Tris, pH 9.0, 7.5 mM MgCl 2 , 200 mM KCl, 1 M methyl glucose, 5 mM dNTPs, 5 U Taq DNA Polymerase, Respectively. In RT-PCR using SYBR Green, an appropriate amount of 10000 × SYBR Green I Nucleic Acid Gel Stain (Cat. No. 50513, Lonza) was added to the reaction composition. In the RT-PCR using the hydrolysis probe analysis method, Probes were used. RT-PCR amplification products were verified in real time using the CFX9600 Real-Time System using SYBR Green or a fluorescent signal from a reporter.
Ct and RFU values for quantitative analysis of fluorescence signal changes were determined using CFX Manager software.
Example One: NSTS Of the probe Experiment to confirm amplification signal enhancement effect by base substitution position
It is an experiment to compare the difference in amplification effect depending on the position of the substituted base on the probe. For this, 1) a basic type NSTS probe (NSTS probe in which cytotoxin (C) is replaced with thymine (T) as the third base from the 5 'end), 2) adenine (A) as the seventh base from the 5' (T), (3) NSTS probe in which adenine (A) as the eighth base from the 5 'end of the basic type NSTS probe is substituted with thymine (T), (4) Ninth base from the 5' end of the basic type NSTS probe NSTS probe in which the adenine (A) is substituted with thymine (T) and ⑤ thymine (T), which is the tenth base from the 5 'end of the basic type NSTS probe, was substituted with adenine (A). The results of RT-PCR experiments using 5 different NSTS probes were compared.
* Test primers and probes
Forward primer: 5'-agcatgtcaagatcacagattttgg-3 '
Rear primer: 5'-gcctccttctgcatggtattctttct-3 '
Basic type NSTS probe ①: 5'-cgTccaaaatctgtgatcttgacat-3 '
Base substituted NSTS probe 2: 5'-cgTccaTaatctgtgatcttgacat-3 '
Base substituted NSTS probe 3: 5'-cgTccaaTatctgtgatcttgacat-3 '
Base substituted NSTS probe ④: 5'-cgTccaaaTtctgtgatcttgacat-3 '
Base substituted NSTS probe 5: 5'-cgTccaaaaActgtgatcttgacat-3 '
* RT-PCR reaction conditions
95 캜, 3 minutes (once)
95 ° C, 15 sec - 62 ° C, 40 sec - 72 ° C, 40 sec (40 repetitions)
* RT-PCR reaction composition
EGFR L858R mutant type plasmid DNA 1 ul (1x10 4 copy) , the two kinds of primers each 1 ul (2 pmol / ul) , the
As shown in FIG. 1, a typical RT-PCR signal in which the SYBR Green signal gradually increases with the generation of the PCR amplification product was observed, and it was confirmed that there was a difference in the amplification efficiency depending on the probe used.
As shown in Table 1, the NSTS probe was found to have a third base substitution from the 5 'terminus (see Table 1). As a result, the Ct value in RT- C-> T), it was confirmed that a form having one of base substitutions of 7th to 10th bases could enhance the PCR amplification signal. Especially, when the position of added substitution base was 8 or 9, PCR amplification was more efficient and Ct value was found to be 5 or more (5.32, 5.86) lower. In addition, the increase of the fluorescence signal (RFU) was confirmed along with the change of the Ct value. (③, ④) with 8th or 9th base substitution in addition to the third base substitution (C-> T) from the 5 'end of the probe showed a fluorescence signal enhancement effect of up to 80%.
NSTS probe
Example 2: NSTS Of the probe Experiment to confirm amplification signal enhancement effect according to the type of 9th bases from 5 'terminal
(C - > T) from the 5 'end of the NSTS probe was compared with that of the ninth base of the base type NSTS probe. To this end, adenine (A), which is the ninth base of the basic type NSTS probe, is substituted with a probe which is not substituted, a probe which is substituted with thymine (T), a probe which is substituted with cystathionine (C) Probe, and four kinds of probes were prepared. The patterns of the RT-PCR amplified signals using the above four probes were compared.
* Test primers and probes
Forward primer: 5'-agcatgtcaagatcacagattttgg-3 '
Rear primer: 5'-gcctccttctgcatggtattctttct-3 '
Basic type NSTS probe ①: 5'-cgTccaaaatctgtgatcttgacat-3 '
Base substituted NSTS probe 2: 5'-cgTccaaaTtctgtgatcttgacat-3 '
Base substituted NSTS probe 3: 5'-cgTccaaaCtctgtgatcttgacat-3 '
Base substituted NSTS probe 4: 5'-cgTccaaaGtctgtgatcttgacat-3 '
* RT-PCR reaction conditions
95 캜, 3 minutes (once)
95 ° C, 15 sec - 62 ° C, 40 sec - 72 ° C, 40 sec (40 repetitions)
* RT-PCR reaction composition
EGFR L858R mutant type plasmid DNA 1 ul (1x10 4 copy) , the two kinds of primers each 1 ul (2 pmol / ul) , the
As shown in FIG. 2 and Table 2, as a PCR amplification product was generated, a typical RT-PCR signal in which the SYBR Green signal gradually increased was observed, and the amplification efficiency was different according to the type of the substitution base of the probe used . (④) substituted with guanidine (G) is highly effective for enhancing the amplification signal of probes (②, ③) in which the ninth base adenine (A) is substituted with thymine (T) or cytosine (C) The signal enhancement effect of
NSTS probe
Example 3: NSTS Of the probe Experiment to confirm amplification signal strengthening effect according to type of 8th bases from 5 'terminal
(C - > T) from the 5 'end of the base type NSTS probe. To this end, adenine (A), which is the eighth base of the basic type NSTS probe, is substituted with a probe which is not substituted, a probe which is substituted with thymine (T), a probe which is substituted with cystathionine (C) Probe, and four kinds of probes were prepared. The patterns of the RT-PCR amplified signals using the above four probes were compared.
* Test primers and probes
Forward primer: 5'-agcatgtcaagatcacagattttgg-3 '
Rear primer: 5'-gcctccttctgcatggtattctttct-3 '
Basic type NSTS probe ①: 5'-cgTccaaaatctgtgatcttgacat-3 '
Base substituted NSTS probe 2: 5'-cgTccaaTatctgtgatcttgacat-3 '
Base substituted NSTS probe 3: 5'-cgTccaaCatctgtgatcttgacat-3 '
Base substituted NSTS probe ④: 5'-cgTccaaGatctgtgatcttgacat-3 '
* RT-PCR reaction conditions
95 캜, 3 minutes (once)
95 ° C, 15 sec - 62 ° C, 40 sec - 72 ° C, 40 sec (40 repetitions)
* RT-PCR reaction composition
EGFR L858R mutant type plasmid DNA 1 ul (1x10 4 copy) , the two kinds of primers each 1 ul (2 pmol / ul) , the
As shown in FIG. 3 and Table 3, as a PCR amplification product was generated, a typical RT-PCR signal in which the SYBR Green signal gradually increased was observed, and there was a difference in amplification efficiency depending on the type of the substitution base of the probe used . (④) substituted with guanidine (G) is highly effective in enhancing the amplification signal of probes (②, ③) in which the eighth base adenine (A) is substituted with thymine (T) or cytosine (C) The signal enhancement effect of
NSTS probe
Example 4: NSTS Of the probe Experiment to confirm amplification signal enhancement effect according to type of 10th base substituted from 5 'end
(C - > T) from the 5 'end of the base type NSTS probe. To this end, thymine (T), which is the tenth base of the NSTS probe, is substituted with a probe which is not substituted, a probe which is substituted with adenine (A), a probe which is substituted with cystathionine (C) , And more than 4 species were produced. RT-PCR amplified signals using the above four probes were compared.
* Test primers and probes
Forward primer: 5'-agcatgtcaagatcacagattttgg-3 '
Rear primer: 5'-gcctccttctgcatggtattctttct-3 '
Basic type NSTS probe ①: 5'-cgTccaaaatctgtgatcttgacat-3 '
Base substituted NSTS probe 2: 5'-cgTccaaaaCctgtgatcttgacat-3 '
Base substituted NSTS probe 3: 5'-cgTccaaaaActgtgatcttgacat-3 '
Base substituted NSTS probe ④: 5'-cgTccaaaaGctgtgatcttgacat-3 '
* RT-PCR reaction conditions
95 캜, 3 minutes (once)
95 ° C, 15 sec - 62 ° C, 40 sec - 72 ° C, 40 sec (40 repetitions)
* RT-PCR reaction composition
EGFR L858R mutant type plasmid DNA 1 ul (1x10 4 copy) , the two kinds of primers each 1 ul (2 pmol / ul) , the
As shown in FIG. 4 and Table 4, as a PCR amplification product was generated, a typical RT-PCR signal in which the SYBR Green signal gradually increased was observed, and the amplification efficiency was different according to the type of the substitution base of the probe used . (④) substituted with guanidine (G) is highly effective for enhancing the amplification signal of probes (②, ③) in which the tenth base thymine (T) is substituted by cytosine (C) or adenine (A) The signal enhancement effect of
NSTS probe
RT-PCR amplification efficiency and amplification signal enhancement effect of NSTS probes on the NSTS probes were found to be highest in Examples 2, 3 and 4, and thymine (T) and cytosine (C) were the largest, followed by adenine (A) and guanidine (G).
T? C? A> G
Example 5: FRET principle applied NSTS Of the probe Base substitution and TaqMan Probe Experiment to confirm amplification signal enhancement effect by addition
RT-PCR using the NSTS probe with the FRET principle was carried out to confirm whether the base substitution enhances the signal amplification as in the case of the test using SYBR Green of Examples 1 to 4. We also tried to confirm the effect of amplifying the amplified signal by using TaqMan probe at the same time. To this end, a 'NSTS probe' in which the third base from the 5 'end of the' general NSTS probe 'was substituted with thymine (T) and the FAM and BHQ1 were bound to both ends, respectively. (NSTS probe in which the third and ninth bases were substituted with thymine (T) from the NSTS probe '[5' terminal, respectively, and FAM and BHQ1 were bound to the two ends of the NSTS probe], and a 'Combo probe' [FAM and BHQ1 ], And hydrolysis probes with structures that can apply the above three FRET principles were prepared, and the amplified signal patterns of RT-PCR using these or each of them were compared.
* Test primers and probes
Forward primer: 5'-ccgcagcatgtcaagatcac-3 '
Rear primer: 5'-gcctccttctgcatggtattctttct-3 '
Normal NSTS probe: 5'-FAM-cgTccaaaatctgtgatcttgac-BHQ1-3 '
T sub. NSTS probe: 5'-FAM-cgTccaaaTtctgtgatcttgac-BHQ1-3 '
Combo probe: 5'-FAM-ctgggtgcggaagagaaagaa-BHQ1-3 '
* RT-PCR reaction conditions
95 캜, 3 minutes (once)
95 캜, 15 sec - 55 캜, 40 sec - 72 캜, 40 sec (repeated 50 times)
* RT-PCR reaction composition
Reaction: 1 μl (1 × 10 4 copy) of EGFR L858R mutant plasmid DNA, 1 μl (10 pmol / μl) of each of the above two primers, 1 μl of normal NSTS probe (15 pmol / μl), and Taq DNA polymerase Were mixed with 4 uL of premixed 5x qPCRMix and sterile water to a final total volume of 20 ul.
Reaction: 1 ul (1 x 10 4 copies) of EGFR L858R mutant plasmid DNA, 1 ul (10 pmol / ul) of each of the two primers, T sub. 1 μl of NSTS probe (15 pmol / μl), and 4 × qPCRMix (5 μ qPCRMix) pre-mixed with the reaction composition required for real-time PCR, such as Taq DNA polymerase, and sterilized water were mixed to a final total volume of 20 μl.
Reaction: 1 μl (1 × 10 4 copy) of EGFR L858R mutant plasmid DNA, 1 μl (10 pmol / μl) of each of the two primers, 1 μl of the Combo probe (15 pmol / μl), 1 μl of the TaqMan probe ) And 4x qPCRMix (5x qPCRMix) pre-mixed with the reaction composition required for real-time PCR, such as Taq DNA polymerase, and sterilized water were mixed to a final total volume of 20 μl.
Reaction: 1 μl (1 × 10 4 copy) of EGFR L858R normal plasmid DNA, 1 μl (10 pmol / μl) of each of the above two primers, T sub. 4 μl of qPCRMix (5 μ qPCRMix) containing 1 μl of NSTS probe (15 pmol / μl), 1 μl of TaqMan probe (10 pmol / μl) and Taq DNA polymerase and the reaction composition required for real-time PCR was mixed with sterilized water, 20 < / RTI >
Reaction (negative control): 1 ul (1 x 10 4 copies) of DDW, 1 ul (10 pmol / ul) of each of the two primers, T sub. 4 μl of qPCRMix (5 μ qPCRMix) containing 1 μl of NSTS probe (15 pmol / μl), 1 μl of TaqMan probe (10 pmol / μl) and Taq DNA polymerase and the reaction composition required for real-time PCR was mixed with sterilized water, 20 < / RTI >
As shown in FIGS. 6 and 5, a typical RT-PCR signal in which the FAM fluorescence signal gradually increases with the generation of the PCR amplification product was observed, and it was confirmed that there was a difference in the amplification efficiency depending on the type of probe used .
In the RT-PCR using the NSTS probe with the FRET principle, the PCR amplification can be effectively performed by base substitution as in the case of the SYBR Green test. That is, it was confirmed that the Ct was lowered by 3.5 or more and the PCR amplified signal (RFU) was also increased by 50% (RFU 2089.44 -> 2953.16).
Using the combo probe with the NSTS probe applied with the FRET principle, the PCR amplification signal was increased by 43% (RFU 2953.16 -> 4256.57).
The results of the reaction and the reaction show that the NSTS probe can accurately distinguish the traits according to SNPs. That is, it was confirmed that nonspecific PCR amplification did not occur.
Example 5 demonstrates that a base-substituted NSTS probe using the FRET principle can accurately distinguish SNP traits without nonspecific reaction, and can be used with a combo probe to amplify the amplified signal to obtain highly sensitive RT-PCR results I could.
NSTS probe
Claims (12)
Wherein the 5'-terminal of the probe is substituted with the polymerase chain reaction product and the non-complementary base, and the 5'-terminal of the probe is 5'-terminal of the polymerase chain reaction product, End of the probe so that the 5'-flap endonuclease activity of the DNA polymerase does not work, and the base of one of the 7th to 10th bases from the 5'-terminal of the probe A method of inspecting a mutant gene by a real-time polymerase chain reaction in which the amplified signal is not matched with the PCR product.
Wherein the DNA polymerase is a heat-resistant DNA polymerase, and wherein the amplified signal is enhanced by a real-time PCR.
A mutant gene to be tested is a real-time PCR-amplified real-time PCR reaction characterized by single point nucleotide polymorphisms, deletion, substitution, and / or insertion of one or more bases .
In order to prevent one base out of the 7th to 10th bases from the 5'-terminal of the probe to match with the polymerase chain reaction product,
Wherein the probe base is selected from the group consisting of thymine (T), cytosine (C), adenine (A) and guanidine (G).
Wherein the probe is a dual-label probe or an unmodified probe in which a reporter pigment and a quencher pigment are simultaneously modified, wherein the amplified signal-enhanced real-time polymerase chain reaction is used to examine the mutant gene.
Wherein the dual label probe in which the reporter pigment and the quencher pigment are simultaneously modified as the probe and the TaqMan probe in which the same reporter pigment is modified as the dual label probe are simultaneously used, How to check genes.
Wherein the probe is substituted with a third base from the 5'-end by a base and a non-complementary base, and the 5'-end of the probe is located at 24-38 bases from the 5'-end of the polymerase chain reaction product , And the base of one of the 7th to 10th bases from the 5'-terminal of the probe is not matched with the polymerase chain reaction product. The kit for real-time polymerase chain reaction for amplifying an amplified signal.
Wherein the mutant gene to be tested comprises single point nucleotide polymorphisms, deletion, substitution and / or introduction of one or more bases, and amplified signal amplified signal amplified gene polymorphism.
Wherein the probe is a dual-label probe or an unmodified probe in which a reporter pigment and a quencher pigment are simultaneously modified.
Wherein the probe comprises a reporter pigment at the 3 'end and a quencher pigment at the 5' end of the forward primer, wherein the amplification signal is enhanced.
Wherein the probe comprises a dual label probe in which a reporter pigment and a quencher pigment are simultaneously modified as the probe and a TaqMan probe in which the same reporter pigment is modified as the dual label probe. Chain reaction kit.
When the non-aqueous probe is used, intercalating agents that bind to DNA double bonds and fluoresce, or surface binding agents that bind to the surface of DNA double bonds and fluoresce, are added Real-time polymerase chain reaction kit for gene mutation testing with enhanced amplification signal.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080118922A1 (en) | 2004-07-23 | 2008-05-22 | National Institute Of Advanced Industrial Science And Technology | Flap Endonuclease Mutants |
| WO2012067828A1 (en) | 2010-11-15 | 2012-05-24 | Exact Sciences Corporation | Mutation detection assay |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080118922A1 (en) | 2004-07-23 | 2008-05-22 | National Institute Of Advanced Industrial Science And Technology | Flap Endonuclease Mutants |
| WO2012067828A1 (en) | 2010-11-15 | 2012-05-24 | Exact Sciences Corporation | Mutation detection assay |
Non-Patent Citations (1)
| Title |
|---|
| Clin. Chem., Vol. 47, No. 11, pp. 2050-2053 (2001.11.) |
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