JP2010246515A - Simultaneous detection of base substitution in deoxyribonucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and quickly performing simultaneous detection of single nucleotide polymorphism (SNP) on a deoxyribonucleic acid (DNA) at a low cost. <P>SOLUTION: A number of SNPs on a DNA are amplified by synthesizing corresponding allele-specific primers, a polymerase chain reaction (PCR) is carried out by using a reaction liquid containing a number of primers, the quantity of the allele-specific primers remaining in the reaction liquid is analyzed by a matrix-assisted laser desorption/ionization-time of flight-mass spectrometer (MALDI-TOF-MS), and the result is compared with quantity before reaction to identify the allele-specific primers used in the reaction and simultaneously determine presence of corresponding base substitution. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a test method for simultaneously detecting a large number of base substitution sites in deoxyribonucleic acid (DNA).

  Single nucleotide substitution polymorphisms (SNPs) on genes are important as a major factor determining the genetic characteristics of individuals, and various testing methods have been developed to detect this nucleotide substitution.

  Numerous allyl-specific primers that can identify SNPs on genes by different chain lengths and fluorescent labeling substances are mixed to perform polymerase chain reaction (PCR), and unreacted allyl-specific primers remaining in the reaction mixture The multiplex SNP test method for checking the presence or absence of the corresponding base substitution by distinguishing by electrophoresis has already been used.

The MassARRAY system is a method for detecting SNPs for the purposes of fine mapping, linkage analysis, and genetic testing by combining primer extension reaction and matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Already used.
Japanese Patent No. 3057553

  The multiplex SNP test method has a problem in that the number of allyl-specific primers required increases as the number of SNP sites to be tested increases, but the cost of the test becomes high because fluorescently labeled primers are expensive.

  Also, in order to identify the difference in primer chain length by electrophoresis, a difference of at least 1 to 3 bases is required, and the migration speed changes depending on the difference in the primer base sequence, so that it can be identified without actually migrating. It is difficult to guess whether it is possible.

め操作が煩雑で，時間と手間がかかるという問題点がある．On the other hand, the primer extension reaction is performed by using unreacted deoxyribonucleic acid (dNTPs), PCR primer, single-stranded DNA from a product obtained by PCR amplification (PrePCR) in advance.

The operation is complicated and takes time and effort.

なるという問題点がある．In addition, it is difficult to control the multiplex PrePCR before the primer extension reaction so that an equal amount of amplification product can be obtained at all sites including the target SNP. Inside MassE for SNP detection

There is a problem that

う問題点がある．Furthermore, in the primer extension reaction, in addition to dNTPs necessary for normal PCR, dideoxyribonucleic acid (ddNTPs), alkaline phosphatase, and SNP detection.

There is a problem.

  Therefore, an object of the present invention is to provide a simultaneous analysis method capable of detecting a large number of SNPs on DNA simply, rapidly and inexpensively by a single PCR.

  The present invention synthesizes an allele-specific primer to amplify a large number of SNPs on DNA, performs PCR, and then determines the amount of allyl-specific primer remaining in the reaction solution as MALDI-TOF-MS. This test method is characterized by identifying allele-specific primers used in the reaction and detecting the presence or absence of corresponding base substitutions at the same time.

  According to the present invention, a large number of SNPs can be tested simultaneously by a single PCR, and the necessary allyl-specific primer does not need to be fluorescently labeled. Because of the high molecular weight measurement accuracy of TOF-MS, even primers with the same chain length can be discriminated from differences in base composition, so it is not necessary to design them so that the chain lengths are different. Can be inspected.

  In addition, no special knowledge is required for primer design, it is very simple without steps such as PrePCR and enzyme treatment, and the peak appearance position can be estimated by calculating the molecular weight. Identification is easy.

  Since the molecular weight range that can be ionized by MALDI-TOF-MS is several thousand to several tens of thousands, a large number of primers with different molecular weights can be simultaneously tested in the range of 10 to 300 oligonucleotides, enabling simultaneous analysis of several hundred to several thousand SNP Become.

  The best mode for carrying out the present invention will be described below.

  Select a SNP according to the purpose, and design a reverse primer that is common to an allele-specific primer having a base substitution site at the 3 'end that opposes the preceding and succeeding base sequences. Calculate the molecular weights of the designed primers for a large number of SNPs, and if the molecular weights are close, add a non-complementary oligonucleotide to the 5 'end to make corrections so that each primer can be easily identified.

  PCR amplification is performed by adding template DNA to a PCR reaction solution containing the synthesized primer mix, dNTPs, polymerase, and the like. Be careful not to overlap the molecular weight of the amplified product with unreacted primers.

  The sample including the control sample is analyzed by MALDI-TOF-MS, the peak of the negative control is compared with the peak of the sample, and the primer used for PCR is identified from the peak in which the decrease is observed.

  If there is a decrease in one or both of the reverse primer and the opposing allele-specific primer, the presence of the corresponding base substitution is observed, and if there is no decrease in both the reverse primer and the allele-specific primer, PCR inhibition Therefore, it is judged that the lack of template DNA and the failure of annealing due to unexpected base substitution occurred.

  Four guanine insertions / deletions (base number 261) in exon 6 of the ABO blood group on human chromosome 6 and cytosine and adenine transversion mutations (base number 796) in exon 7 An allele-specific primer and two reverse primers were synthesized.

  DNA was extracted from blood with a known ABO blood group, mixed with a PCR reaction solution containing the synthesized primer mix, and PCR was performed with a thermal cycler.

  The solution after PCR was desalted, mixed with the matrix on the plate and dried, and analyzed by MALDI-TOF-MS.

  Compared with the negative control, a decrease in the molecular weight peak corresponding to the specific primer was observed, and the primer used for PCR could be identified.

  From the identified primers, the presence or absence of the corresponding base substitution was specified, and ABO blood group genotyping was performed.

Claims (1)

  Allyl-specific primers are synthesized to amplify a number of single nucleotide substitution polymorphisms (SNPs) on deoxyribonucleic acid (DNA) and remain in the reaction mixture after polymerase chain reaction (PCR). The amount of allyl-specific primer is analyzed by a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and compared with the amount before reaction to identify the allyl-specific primer used in the reaction. The method of checking for the presence of corresponding base substitutions simultaneously.