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WO2006031061A1 - Procede de selection de sonde du gene fret utilisant le criblage par pcr - Google Patents

Procede de selection de sonde du gene fret utilisant le criblage par pcr Download PDF

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Publication number
WO2006031061A1
WO2006031061A1 PCT/KR2005/003038 KR2005003038W WO2006031061A1 WO 2006031061 A1 WO2006031061 A1 WO 2006031061A1 KR 2005003038 W KR2005003038 W KR 2005003038W WO 2006031061 A1 WO2006031061 A1 WO 2006031061A1
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WIPO (PCT)
Prior art keywords
gene
primers
target gene
target
strand
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PCT/KR2005/003038
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English (en)
Inventor
Nam Woong Song
Hyong-Ha Kim
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Korea Research Institute Of Standards And Science
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Publication of WO2006031061A1 publication Critical patent/WO2006031061A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention relates to a method for screening a gene detection probe set using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • a rapid detection system for a certain gene, based on DNA amplification, is necessary for the diagnosis of various diseases or the detection of genetically modified food.
  • the existing systems for the detection of target DNA require the amplification of target DNA by PCR and/or the culture of microorganisms having target DNA. Since PCR is based on the sequence- specific hybridization of two primers to template DNA, the amplification by PCR theo ⁇ retically allows the sequence-specific detection of target DNA.
  • sequence-specific PCR has a fundamental problem in that it fails to amplify because the amplication of byproduct or PCR is not an enzyme reaction conforming to a strict stoichiometry. Another problem is that it is difficult to use in the quantification of a target gene (Biosensors & Bioelectronics 14 (1999) 401-408).
  • the FRET (fluorescence resonance energy transfer) system uses a process of transferring fluorescent energy while inducing resonance interaction between two chromophores, called an "energy donor” and an “nergy acceptor”, and is based on the Forster's theory explaining that energy transfer efficiency is in inverse proportion to the 6 th power of the distance between the energy donor and acceptor (Forster theory; J.R. Lakowicz. Principles of Fluorescence Spectroscopy, Plenum Press, New York and London, 1983).
  • n refractive index of medium
  • R is a distance where a possibility for the donor energy to be transferred to the acceptor is 50%, and it has a value in a range of 3-4 nm, depending on a combination of the energy donor and the energy acceptor.
  • a change in distance in a range of about ViR -2R can be experimentally measured.
  • This principle of the fluorescence resonance energy transfer can be used in methods of analyzing the base sequence of a certain gene.
  • the two chromophores corresponding to the energy donor and the energy acceptor, respectively must be located in a very short distance, i.e., in a range of 3-4 nm. Accordingly, this principle can be effectively used in a process of screening and quantifying a gene with a certain base sequence in a given solution.
  • the energy donor and energy acceptor which freely float in a solution do not emit the FRET fluorescence because the average distance therebetween is long.
  • an oligomer (a kind of primer) labeled with the energy donor and acceptor is hybridized to a target gene (DNA or RNA) containing a certain base sequence, the FRET fluorescence will be emitted because two probes become closer through their binding to the gene, as shown in FIG. 1.
  • the fluorescence resonance energy transfer occurs as described above, the fluorescent spectrum of the gene will be changed, resulting in an actual change in the color of the solution.
  • the method of inducing FRET fluorescence by two probes and observing a change in fluorescent spectrum is also called “dual probe color change- FRET"(hereinafter, referred to as "DPCC-FRET'.
  • DPCC-FRET dual probe color change- FRET
  • two in ⁇ dependent probes corresponding to the donor and the acceptor are used and consist of the same base number (n), and thus, only when base sequences of 2n for the two probes complementarity match with target gene, the FRET phenomenon will appear.
  • the method for detecting a gene using DPCC-FRET has the following advantages.
  • the method since it is possible to make the gene probe length (the number of bases in sequences) short, the method has excellent ability to identify the mismatch in base sequence between a target gene and gene probes. If the length of probes becomes longer, the ability to identify the mismatch in base sequences therebetween will be weaken, because the hybridization will easily occur as shown in FIG. 2 even when not all the base sequences match between the probe and target gene.
  • the two gene probes should all be hybridized to the target gene to detect the
  • the target gene can be detected with the same scarcity or se ⁇ lectivity in base sequence as gene probes containing two times longer gene sequences.
  • DNA/RNA or new substance peptide nucleic acid (PNA) with two fluorescent substances corresponding to the energy donor and acceptor, respectively, are mainly used as a set of gene probes, and the fluorescence-labeled DNA/RNA or PNA oligomer is purchased with many costs. Particularly, because the PNA oilgomer requires many production costs even in an unlabeled state, exorbitant cost is required to purchase the fluorescence-labeled PNA oligomer.
  • Another object of the present invention is to provide a method for screening a probe set for detecting a foreign gene introduced into animals/plants, using the FRET system.
  • Still another object of the present invention is to provide a method for screening a probe set for detecting an herbicide-resistant gene EPSPS introduced into plants, using the FRET system.
  • Yet another object of the present invention is to provide a method for detecting a gene using said gene detection probe set by the FRET system.
  • the present invention provides a method for screening a probe set for detecting a target gene using the FRET system.
  • the present invention provides a method for screening a probe set for detecting a target gene using the FRET system, the method comprising the steps of: (A) determining one common primer to a complementary strand from the base sequence of a target gene and designing a plurality of different primers to a target strand; (B) mixing the primers with DNA containing the target gene, and subjecting the mixtures to PCR so as to first screen primers to the target strand, which produce products; (C) mixing the first screened primers with one common primer to the com ⁇ plementary strand and DNA containing no target gene, subjecting the mixtures to PCR so as to second screen primers to the target strand, which produce no product; and (D) selecting any two primers from the group consisting of the second screened primers to the target strand and labeling the two selected primers with an energy donor and energy acceptor for FRET test, respectively.
  • the two primers selected from the second screened primers to the target strand are located so close that a change in fluorescent spectrum occurs by the FRET system.
  • the interval between the 3 -terminal end of a primer bound to the forward portion of the target gene and the 5 -terminal end of a primer bound to the reverse portion of the target gene will preferably be 1-8 nm (an interval of 3-24 bases), and more preferably 3-4 nm (an interval of 9-12 bases).
  • the present invention provides a method for detecting a target gene using the
  • the primer set two primers, i.e., probes
  • the energy donor and acceptor bound to the probes become closer to each other within a given distance while emitting fluorescence upon irradiation with laser light.
  • the measurement of a change in fluorescent spectrum can determine if the target gene exists in the DNA.
  • the target gene is described for convenience in the present invention, it is to be understood that the definition of the target gene is not limited only to genes, and any DNA or mRNA having a certain sequence can also be detected according to the present invention.
  • the target gene may be selected from various kinds of genes. For example, genes showing pathogenicity, genes inducing mutation or genes introduced for the transformation of animals and plants may be included in the definition of the target gene. More specifically, according to the present invention, it is also possible to screen a probe set for detecting herbicide-resistant gene EPSPS introduced into plants. In this case, the probe set may be selected from primers forth in SEQ ID NOS: 1, 2, 3, and 5 to 11.
  • the inventive method for screening the gene detection probe set can be effectively used in the expensive DPCC-FRET labeling method, since only the primers selected by the screening of the primer sequences are used after treatment (labeling of the energy donor and acceptor as described below). Particularly, the method of estimating the binding efficiency of primers using polymerase chain reaction is cost-effective as compared to a case where a gene probe set labeled by the DPCC-FRET system is directly used.
  • the present invention provides a target gene detection method which allows whether a target gene exists in a certain DNA to be determined in a simple and easy manner, the method comprising mixing the target gene detection probe set selected by the above-described method with a certain DNA and subjecting the mixture to PCR.
  • the present invention provides a method for screening a target gene, which comprises mixing the target gene detection probe set selected by the above method with a certain DNA isolated from a sample and measuring a change in the fluorescent spectrum of the mixture to determine if a target gene exists in the DNA.
  • a gene detection probe set capable of inducing a false-positive signal can be detected in advance.
  • a primer set efficient for the detection of the gene B is first screened by polymerase chain reaction and is then used to perform PCR reaction for another gene (e.g., gene A) to confirm that it shows no amplification action for the another gene, whether the signal of the gene A, which is not to be generated, can occur, can be determined in advance by polymerase chain reaction.
  • another gene e.g., gene A
  • DNA chips can be effectively selected.
  • a method for detecting a target gene using a gene and probes fixed onto a DNA chip still has a problem in that a false-positive signal and a false-negative signal cannot be distinguished from each other due to various factors. For this reason, whether the gene detection probes properly bind to the target gene needs to be determined in advance by a method for analyzing the false-positive and false-negative signals.
  • the present invention provides a method for screening gene detection probes by observing gene products produced by polymerase chain reaction and estimating the binding efficiency of detection probes.
  • one common primer to a complementary strand on DNA is used and a plurality of different primers to a target strand are designed and used. If all these primers are used to perform polymerase chain reaction and gene products obtained by this PCR reaction are analyzed by agarose gel electrophoresis, results as shown in FIG. 4 can be obtained. Primers corresponding to sequences well amplified in the polymerase chain reaction are determined to have a high efficiency of binding to a target gene (first screening of primers).
  • the gene detection probes screened by the above- described procedures were labeled with fluorescence by the DPCC-FRET system and used to detect a target gene in DNA, and the results are shown in FIG. 6. As can be seen in FIG. 6, if the target gene DNA is contained in a sample, the fluorescent spectrum will change by the corresponding gene probes. Accordingly, in DPCC-FRET fluorescence tests, the screening method according to the present invention can be ef ⁇ fectively used to design and develop the optimal gene detection probe set.
  • the inventive screening method whether primers have specific selectivity to a gene containing a certain base sequence can be determined in advance. Namely, the first screened primers to a target strand, the one common primer to a complementary strand, and (1) DNA containing a target gene or (2) DNA containing no target gene, were mixed with each other and subjected to a conventional polymerase chain reaction. As a result, as shown in FIG. 5, the resulting products were different between the case of DNA containing a target gene and the case of DNA containing no target gene. Namely, it could be seen that the first screened primers (SEQ ID NO: 4 was out of the question because it was excluded in the first selection) could act specifically only on the target gene to produce PCR products.
  • PCR products are produced by a non-target gene in a large amount, it can be seen that the gene detection primers well bind to the non-target gene.
  • the probe set has high efficiency for amplifying the target gene, the probe set is determined to be unsuitable for the detection of the target gene (second screening).
  • FIG. 1 is a conceptual diagram showing a principle for detecting the base sequence of a certain gene using a fluorescence resonance energy transfer (FRET) labeling method.
  • FRET fluorescence resonance energy transfer
  • FIG. 2 is a conceptual diagram showing possible hybrids if there is a mismatch in base sequence between primers and a target gene.
  • FIG. 3 is a conceptual diagram showing the inventive primer design process used in polymerase chain reaction for testing the binding efficiency of gene detection probes.
  • FIG. 4 is a photograph showing electrophoresis results for PCR products obtained by the inventive method.
  • FIG. 5 is a photograph showing electrophoresis results for the PCR products of genes containing the base sequence of a target gene and the PCR products of genes containing no base sequence of a target gene.
  • FlG. 6 is a graphic diagram showing results for the detection of a target gene by the use of gene detection probes obtained by the inventive screening method and labeled by the fluorescence resonance energy transfer method.
  • Example 1 Screening of gene probes containing certain base sequence
  • primers capable of amplifying a fragment of about 2 kbp corresponding to the EPSPS gene were used as the forward primer, and NOS3R (25mer; ATG-
  • TATAATTGCGGGACTCTAATCA (SEQ ID NO: 14) was used as the reverse primer.
  • PCR reaction was performed in gradient cycler PTC-0225 set to the following PCR conditions: denaturation at 94°C for 5 min; amplification of 35 cycles each consisting of 20 sec at 94°C, 30 sec at 45°C, and 2 min at 72°C; and final elongation at 72°C for 10 min.
  • polymerase chain reaction for a primer corresponding to SEQ ID NO: 4 was not well made. Accordingly, this primer can be estimated to be low in the efficiency of binding to a target gene and can be determined to be unsuitable as a sequence for detecting a target gene using the FRET method. Namely, SEQ ID NO: 4 was excluded in the first screening.
  • Example 2 Increase of selectivity to gene containing certain base sequence
  • a chromosome containing the base sequence of the target gene and (2) a chromosome containing no target gene were subjected to polymerase chain reaction in the same conditions as in Example 1 using primers to be used as gene probes. By this process, it could be determined if primers to be used as gene probes have the property of binding only to the target gene.
  • the target gene used was genomic DNA extracted from GM soybean, and the gene having no connection with the target gene was genomic DNA extracted from nonGM soybean.
  • Example 3 Application of gene probes screened by inventive screening method
  • the DPCC-FRET probes were constructed by a process where a probe set designed only with the base sequence of a target gene was first screened by polymerase chain reaction and then labeled with fluorescent substances. Namely, genomic DNA extracted from GM soybean was used as a target protein, and four sets of target gene detection PNA probes selected from the primers for target gene, which have been prescreened by the methods described in Examples 1 and 2, were used (Table 2). The used probes lacked some of bases at the 5 and/or 3-terminal ends of the relevant sequences shown in FIG. 2. This means that these probes have the omission of about 3 in base number due to a problem in PNA construction technology and can be used as probes even in this state, and this possibility was verified in advance.
  • Rh which serve as an energy donor and an energy acceptor, respectively.
  • R binds to the 3-terminal end of the probes, and Rh binds to the 5-terminal end of the probes.
  • the PNA probes were constructed using 5-carboxyfluorescein, succinimidyl ester for the introduction of R (energy donor) and 5-carboxytetramethylrhodamine, succinimidyl ester for the introduction of Rh (energy acceptor). 12 ml of each of the constructed probe sets was used alone or in a mixture with 12 ml of DNA containing a target gene, and allowed to react in the following conditions for 24 hours. Then, fluorescence emission intensity of the reaction solution was measured using a FRET signal detector (see Journal of Photoscience 11(2), 47-53, (2004)) manufactured by the present inventors (FIG. 6).
  • test tubes are prepared: a test tube containing only fluorescence detector reagents (donor concentration: 1 ⁇ M, and acceptor concentration: 3 ⁇ M); and two test tubes each containing a mixture of a target gene (concentration: 1,250 ng/mL) and fluorescence detection reagents (donor concentration: 1 ⁇ M, and acceptor con ⁇ centration: 3 ⁇ M) mixed at a volume ratio of 1:1:1 or 12:12:12 (DNA: donor: acceptor).
  • a CCD light detector mounted on the output stage of a spectrograph with a focus distance of 150 mm, which is equipped with a grating of 150 grooves/mm, is used to observe spectra in a range of 280-850 nm at the same time.
  • the sample solution is irradiated with laser light for 30 seconds while emitted fluorescence is measured two times, and then, the average of the measured values is calculated and recorded.
  • FIG. 6 shows a test result for probe set B, the same result was also shown for other probe sets (data not shown).
  • the detection of a target gene can be performed by DPCC-FRET in an easy and cost-effective manner. Namely, it was demonstrated that the inventive method for screening gene probes using polymerase chain reaction could be effectively used to design and develop the optimal gene probes.
  • the inventive screening method not only allows effective detection of a target gene but also can increase the accuracy and selectivity of probes to a target gene and can greatly reduce the cost for the development of the probes.

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Abstract

L'invention concerne un procédé de criblage d'un ensemble de sondes pour détecter un gène cible au moyen d'un système FRET. Ledit procédé consiste: (A) à déterminer une amorce commune à un brin complémentaire de la séquence de base d'un gène cible et à concevoir plusieurs amorces différentes au brin cible; (B) à mélanger les amorces avec l'ADN contenant le gène cible et à soumettre les mélanges à la PCR de façon à cribler d'abord les amorces au brin cible, ce qui permet de réaliser des produits; (C) à mélanger les premières amorces criblées à une amorce commune au brin complémentaire et l'ADN ne contenant aucun gène cible, à soumettre les mélanges à la PCR de façon à cribler ensuite les amorces au brin cible, aucun produit n'étant alors réalisé; et (D) à choisir deux amorces quelconques du groupe formé des secondes amorces criblées au brin cible et à étiqueter les amorces choisies avec un donneur et un récepteur d'énergie pour l'essai FRET, respectivement. L'utilisation de ce procédé permet non seulement de réduire les dépenses considérables que nécessite le processus d'étiquetage génique, mais aussi d'augmenter la précision et la sélectivité d'un ensemble de sondes vers un gène cible de façon à effectuer plus rapidement et plus facilement un procédé de détection génique.
PCT/KR2005/003038 2004-09-18 2005-09-14 Procede de selection de sonde du gene fret utilisant le criblage par pcr WO2006031061A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293962A (zh) * 2014-10-20 2015-01-21 苏州大学 一种筛选公共引物的方法
CN109402236A (zh) * 2018-11-12 2019-03-01 长江大学 一种水稻育种材料中抗稻瘟病基因Pi-25的检测方法

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KR20010100216A (ko) * 2000-03-16 2001-11-14 박한오 유전자 변형 농산물 검출방법 및 검출용 프라이머
WO2003068964A1 (fr) * 2002-02-15 2003-08-21 Nisshin Seifun Group Inc. Procede permettant de tester les aliments
KR20040012260A (ko) * 2002-08-02 2004-02-11 (주)바이오니아 유전자변형농산물에 대한 유전자변형농산물 혼입률(%)의정량분석방법

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WO2003068964A1 (fr) * 2002-02-15 2003-08-21 Nisshin Seifun Group Inc. Procede permettant de tester les aliments
KR20040012260A (ko) * 2002-08-02 2004-02-11 (주)바이오니아 유전자변형농산물에 대한 유전자변형농산물 혼입률(%)의정량분석방법

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KARADAG A. ET AL: "A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome", NUCLEIC ACIDS RESEARCH, vol. 32, no. 7, 19 April 2004 (2004-04-19), pages 63 *
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VAITILINGOM M. ET AL: "Real-time quantitative PCR detection of genetically modified maximizer Maize and roundup ready soybean in some representative foods", J. AGRIC. FDDO CHEM., vol. 47, no. 12, 1999, pages 5261 - 5266 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293962A (zh) * 2014-10-20 2015-01-21 苏州大学 一种筛选公共引物的方法
CN109402236A (zh) * 2018-11-12 2019-03-01 长江大学 一种水稻育种材料中抗稻瘟病基因Pi-25的检测方法

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