Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to a method for detecting a mutant gene of Perilla island amyloid protein, and a nucleic acid probe and a kit therefor.
• Islet Amyloid Polypeptide is a major component of amyloid that is frequently deposited on the knee island of type 2 diabetic patients, and is secreted into the blood together with insulin from Teng; 3 cells. Have been.
• a missense mutation S20G mutation in which the serine at position 20 of the IAPP amino acid sequence is replaced with glycine is present in about 2.6% of Japanese type 2 diabetes patients, of which about 10% of younger onset patients. It is said that the presence of this mutation increases the risk of developing diabetes.
• IAPP S20G mutation a restriction enzyme recognition site appears in that part, and amplification is carried out by PCR to include the mutated part. It is known that detection is carried out by a method (PCR-RFLP) that detects whether the DNA fragment has been cleaved by electrophoresis (PCR-RFLP). Do, P. 56-59).
• PCR amplifies several billion times more molecules from a few type II molecules, so even a small amount of the amplification product can cause false positives and false negatives.
• PCR-RFLP requires removal of the amplification product after PCR reaction and treatment with restriction enzymes, the amplification product may be mixed into the next reaction system. Thus, false-positive and false-negative results may be obtained.
• treatment with restriction enzymes is performed, and then electrophoresis is performed, so that the time required for detection is extremely long. In addition, automation is difficult due to the complicated operation.
• An object of the present invention is to specify a quenching probe effective for detecting the IAPP S20G mutation, and to provide a method for detecting the IAPP S20G mutation and a kit therefor.
• the present inventors have found that by designing a quenching probe based on a specific region containing the IAPP S20G mutation, it is possible to detect the IAPP S20G mutation by melting curve analysis using the quenching probe, and completed the present invention.
• the present invention provides the following.
• a nucleic acid probe whose end is labeled with a fluorescent dye and whose fluorescence decreases when hybridized, 13 to 30 bases ending with base number 247 in the base sequence shown in SEQ ID NO: 1
• the above nucleic acid probe which has a sequence complementary to the long sequence and has a 5 ′ end labeled with a fluorescent dye.
• nucleic acid probe according to (1) wherein the nucleic acid probe has SEQ ID NO: 12 or 13.
• nucleic acids having single nucleotide polymorphism sites perform melting curve analysis by measuring the fluorescence of the fluorescent dye using a nucleic acid probe labeled with a fluorescent dye, and based on the results of the melting curve analysis.
• a single nucleotide polymorphism The nucleic acid encoding the Ujishima amyloid protein is a mutation in the nucleotide sequence that causes a mutation in which the serine at position 20 of the amino acid sequence of the perilla islet amyloid protein is replaced with glycine.
• the kit for the method according to (3) which comprises the nucleic acid probe having a sequence complementary to the sequence of (5) and having a terminal labeled with a fluorescent dye.
• a region containing a nucleotide sequence mutation that results in a mutation in which the serine at position 20 of the amino acid sequence of Perilla islet amyloid protein is replaced with glycine in the nucleic acid encoding the Kura-jima island amyloid protein is treated with DNA polymerase.
• FIG. 1 shows the position of the quenching probe where the mutation is indistinguishable.
• FIG. 2 shows the location of the quenching probe that can identify the mutation.
• FIG. 3 shows the sensitivity of the method of Example 1 with respect to the absolute amount of genomic DNA.
• FIG. 4 shows the reproducibility of the method of Example 1.
• FIG. 5 shows the detection sensitivity and the quantification of the ratio of mutants in the method of Example 1.
• the probe of the present invention is a nucleic acid probe whose end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye is reduced upon hybridization, wherein the base represented by SEQ ID NO: 1 It has a complementary sequence to a sequence having a length of 13 to 30 bases ending with base number 247 in the sequence, and is characterized in that the 5 'end is labeled with a fluorescent dye.
• a complementary sequence means that it is complementary to the entire length of the subject sequence.
• the probe of the present invention has a structure similar to that of the nucleotide sequence shown in SEQ ID NO: 1 (a sequence having a mutated nucleotide in the IAPP S20G mutation) which has a sequence complementary to a sequence of 13 to 30 nucleotides in length ending at base number 247. It may be the same as the quenching probe described in Reference 1. Examples of the base sequence of the quenching probe used in the present invention include SEQ ID NOS: 12 and 13. As the fluorescent dye, those described in Patent Document 1 can be used. Specific examples include FAM (trademark), TAMRA (trademark), B0DIPY (trademark) FL, and the like.
• the method for binding the fluorescent dye to the oligonucleotide can be performed according to a conventional method, for example, the method described in Patent Document 1.
• a nucleic acid having a single nucleotide polymorphism site is subjected to melting curve analysis by measuring the fluorescence of a fluorescent dye using a nucleic acid probe labeled with a fluorescent dye, and the results of the melting curve analysis
• a single nucleotide polymorphism, wherein the serine at position 20 of the amino acid sequence of Tengla islet amyloid protein is replaced with glycine in the nucleic acid encoding the knee islet amyloid protein It is a mutation in the nucleotide sequence that causes a mutation, and the nucleic acid probe is a probe of the present invention.
• the detection method of the present invention comprises the steps of amplifying a region containing the IAPP S20G mutation of DNA encoding IAPP, and using the probe of the present invention, except for ordinary nucleic acid amplification and melting curve analysis (Tm analysis). Can be performed according to the method described in
• a method using a polymerase As a method for nucleic acid amplification, a method using a polymerase is preferable, and examples thereof include PCR, ICAN, and LAMP.
• amplification is performed by a method using a polymerase, it is preferable to perform amplification in the presence of the probe of the present invention. It is easy for those skilled in the art to adjust the amplification reaction conditions and the like according to the probe used. As a result, only the Tm of the probe is analyzed after amplification of the nucleic acid, and there is no need to handle the amplified product after the reaction. Therefore, there is no risk of contamination by the amplification products. In addition, it is possible to detect with the same equipment as the equipment necessary for amplification, so it is necessary to move the container. I don't. Therefore, automation is easy.
• the primer pair used for PCR can be set in the same manner as the method for setting a primer pair in ordinary PCR, except that a region where the probe of the present invention can be hybridized is amplified.
• the length and Tm of the primer are usually 40 to 70 ° C for 10 to 40 mer, preferably 55 to 60 ° C for 15 to 25 mer.
• the length of each primer in the primer pair may not be the same, but it is preferable that the Tm of both primers is almost the same (usually, the difference is within 2 ° C).
• the Tm value is a value calculated by the nearest neighbor method. Examples of the primer pair include those comprising a primer having the nucleotide sequence shown in SEQ ID NOS: 2 and 3.
• PCR is preferably performed in the presence of the probe of the present invention used in the present invention.
• Tm analysis can be performed without performing an operation for handling the amplification product after the completion of the amplification reaction. It is easy for those skilled in the art to adjust the Tm of the primer and the reaction conditions for PCR according to the probe used.
• composition of a typical PCR reaction solution is as follows.
• Tm analysis can be performed according to a usual method except for measuring the fluorescence of the fluorescent dye of the probe of the present invention. Fluorescence can be measured by measuring light having an emission wavelength using excitation light having a wavelength corresponding to the fluorescent dye.
• the heating rate in Tm analysis is usually 0.1 to 1 ° C / sec.
• the composition of the reaction solution when performing Tm analysis is not particularly limited as long as hybridization between the probe and a nucleic acid having a sequence complementary to its base sequence is possible.
• the cation concentration is 1.5-5 raM and the pH is 7-9. Since the reaction solution of the amplification method using a DNA polymerase such as PCR usually satisfies these conditions, the reaction solution after amplification can be used for Tm analysis as it is.
• Detection of the IAPP S20G mutation based on the result of Tm analysis can be performed according to a conventional method.
• the detection in the present invention includes not only detection of the presence or absence of mutation but also quantification of mutant DNA and measurement of the ratio of wild-type DNA to mutant DNA.
• the kit of the present invention is a kit for use in the detection method of the present invention.
• This kit is a nucleic acid probe (quenching probe) whose end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases upon hybridization, and ends with base number 247 in the base sequence shown in SEQ ID NO: 1. It comprises a nucleic acid probe having a sequence complementary to a sequence having a length of 13 to 30 bases and having a 5 ′ end labeled with a fluorescent dye.
• the quenching probe is as described above with respect to the probe of the present invention.
• the detection kit of the present invention may further include, in addition to the quenching probe, reagents required for performing nucleic acid amplification in the detection method of the present invention, in particular, primers for amplification using DNA polymerase.
• the quenching probe, the primer and other reagents may be housed separately, or a part of them may be a mixture.
• the primers shown in Table 2 were designed to amplify the portion containing the S20G mutation.
• the position indicates the base number in the base sequence shown in SEQ ID NO: 1.
• PCR and Tm analysis were performed using the Smart Cycler System (Cephied) under the following conditions.
• the excitation wavelength and the detection wavelength in Tm analysis were 450 to 495 nm and 505 to 537 nra (BODIPY FL), 527 to 555 nm and 565 to 605 nm (TAMRA), respectively.
• Table 4 The excitation wavelength and the detection wavelength in Tm analysis were 450 to 495 nm and 505 to 537 nra (BODIPY FL), 527 to 555 nm and 565 to 605 nm (TAMRA), respectively.
• Tm analysis (l ° C / sec) As a result of PCR and Tm analysis using each probe, probe 5FL-mt- 1-18, 5T-mt-1-18, 5FL_mt- 1-21, and 5T-mt- Only when 1-21 was used, a change in fluorescence intensity that could be analyzed by Tm analysis was observed.
• the arrangement of each probe with respect to the nucleotide sequence containing the IAPP S20G mutation is shown in FIGS. 1 and 2.
• the wild-type sequence (SEQ ID NO: 15) and the mutant sequence (SEQ ID NO: 16) correspond to base numbers 213 to 262 of the base sequence of SEQ ID NO: 1.
• F indicates a fluorescent dye.
• whether the probe can be used in Tm analysis is considered to depend on the position of C to which the fluorescent dye is attached, and the length of the probe is limited as long as it includes the polymorphic site. Not very important.
• a plasmid having a wild-type base sequence (same as the above plasmid except that base number 285 is A in the base sequence of SEQ ID NO: 1) was prepared.
• Ten samples (wt / rat) were prepared by mixing wild-type plasmid and this mutant-type plasmid, and a sample containing only wild-type plasmid (wt / wt) and a sample containing only mutant-type plasmid (mt / rat) were prepared. / m
• Fig. 4 shows the results. As is clear from FIG. 4, the method was excellent in reproducibility.
• Fig. 5 shows the results. The height of both peaks changed according to the ratio, and it was shown that the ratio could be determined based on the ratio of the heights of both peaks.
• the vertical axis represents the value of the inverse sign of the first derivative of the fluorescence intensity ( ⁇ dF / dt).
• the horizontal axis represents the temperature (° C.).
• a quenching probe effective for detecting an IAPP S20G mutation is provided, and a method for detecting an IAPP S20G mutation using the same and a kit therefor are provided. Since Tm analysis is completed in tens of seconds, the time required for detection can be greatly reduced. According to a preferred embodiment of the present invention in which amplification of a nucleic acid in the presence of a probe is combined with Tm analysis, it is only necessary to analyze the Tm of the probe after amplification of the nucleic acid, so that there is no need to handle the amplification product after the reaction is completed. Therefore, there is no need to worry about contamination by amplification products. In addition, since the detection can be performed with the same device as that required for amplification, it is not necessary to move the container. Therefore, automation is easy.

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• 2003
  • 2003-04-18 JP JP2003114380A patent/JP4454249B2/ja not_active Expired - Fee Related
• 2004
  • 2004-04-16 AT AT04728056T patent/ATE428782T1/de not_active IP Right Cessation
  • 2004-04-16 US US10/553,614 patent/US7332306B2/en not_active Expired - Fee Related
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