Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism

a technology of nucleic acids and reagents, applied in the field of amplifying nucleic acids, can solve the problems of significant amplification of byproducts, and achieve the effect of not amplifying or inhibiting the amplification of the desired nucleic acid

Inactive Publication Date: 2005-11-24
AISIN COSMOS R&D +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes how to use a special kit to quickly and accurately detect small differences between genetic sequences called Single Nucleotide Polymorphisms (SNPs). This method involves creating a specific mixture of ingredients that includes enzymes, molecules used for building new DNA, and proteins needed for this process. When all these components are mixed together with a sample containing the target DNA, they form a reaction solution where the DNA polymerase helps build new DNA based on the instructions provided by the original DNA. If there is no complete match between the two templates, then the resulting DNA will have a different pattern than what was expected. Using this technique makes it easy to identify SNPs without having to manually analyze them yourself.

Problems solved by technology

The technical problem addressed in this patent is how to effectively amplify a desired nucleic acid without producing large amounts of non-specific byproducts during PCR amplification. This applies particularly to detection of single nucleotide polymorphisms where it can be difficult to distinguish between the desired mutation and other variants that might occur naturally or through contamination.

Method used

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  • Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism
  • Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism
  • Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism

Examples

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example 1

[0133] A human genome DNA (Promega) was prepared as a template DNA as shown in FIG. 1. Further, 16 kinds of oligonucleotides (Oligonucleotides 1 to 16) were prepared as the primer DNAs. Each primer DNA was designed with reference to Homo sapiens PAC clone from RP5-852P6 from 7p11.2-p21, complete sequence (Genbank accession no.; AC006454). The Genbank accession no.; number refers to an access number of Gene Bank (the same in the following). Since each primer DNA has possibility of any primer design, each of the positions was shifted to design the primer DNA. Each primer DNA consists of a 20mer base sequence which is 100% complementary to the template DNA. Each primer DNA may be synthesized by a known method on the basis of the base sequence of the template DNA.

Oligonucleotide 1:5′-ggtgcactcc atcatgctta-3′Oligonucleotide 2:5′-catcagt cagaggggct cac-3′Oligonucleotide 3:5′-cccacatccc tggcaggaat-3′Oligonucleotide 4:5′-tgcaggt gtgggcctag ctg-3′Oligonucleotide 5:5′-tgtcctgggc cccagcagga-...

example 2

[0177] Next, Example 2 will be explained. Explanation of the parts which are similar to those of the Example 1 will be omitted or simplified.

[0178] In this Example, as shown in FIG. 3, a human genome DNA was prepared as a template DNA, and 12 kinds of oligonucleotides (Oligonucleotides 17 to 28) were prepared as the primer DNAs. Each primer DNA was designed with reference to Homo sapiens BAC clone from RP11-16P10 from 7, complete sequence (Genbank accession no.; AC093734, AC011786). Since each primer DNA has possibility of any primer design, each position was shifted to design the primer DNA. Each primer DNA consists of a 20mer base sequence which is 100% complementary to the template DNA.

Oligonucleotide 17:5′-cgggtgc acacaaaggc tgg-3′Oligonucleotide 18:5′-tctctggcca ggtgcctggc-3′Oligonucleotide 19:5′-cgccccg acaaccctga ccc-3′Oligonucleotide 20:5′-cttgggaaga tcctgagact-3′Oligonucleotide 21:5′-tcggtaa acgctggctc ccg-3′Oligonucleotide 22:5′-caaaacgccc cccaccgccc-3′Oligonucleotide 2...

example 3

[0196] Next, Example 3 will be explained. Explanation of the parts which are similar to those of each of the above-mentioned Examples will be omitted or simplified.

[0197] As shown in FIG. 5 and FIG. 6, a human genome DNA was prepared as a template DNA, and 8 kinds of oligonucleotides (Oligonucleotides 29 to 36) were prepared as the primer DNAs. Each primer DNA was designed with reference to the Human DNA sequence from clone RP5-1013A22 on chromosome 20 Contains the HNF4A (hepatic nuclear factor 4, alpha) gene, part of a novel gene encoding a protein similar to cellular retinaldehyde-binding protein, a RPL37A (ribosomal protein L37a) pseudogene, parts of 2 novel genes, ESTs, STSs and GSSs, complete sequence (Genbank accession no.; AL132772). Further, Oligonucleotide 31 and Oligonucleotide 32 were designed with reference to Homo sapiens 3q BAC RP11-529F4 (Roswell Park Cancer Institute Human BAC Library) complete sequence (Genbank accession no.; AC080007). Further, Oligonucleotide 33 ...

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Abstract

An object of the present invention is to provide a nucleic acid amplification method for amplifying a desired nucleic acid while suppressing amplification of byproducts in a PCR reaction, a reagent kit used for nucleic acid amplification, a method of detecting single nucleotide polymorphism to detect single nucleotide polymorphism by utilizing that amplification of byproducts is suppressed in a PCR reaction, and a reagent kit used for detecting single nucleotide polymorphism. The method of amplifying nucleic acids by PCR is characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from Thermus thermophilus, and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.

Description

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Claims

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Application Information

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Owner AISIN COSMOS R&D
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