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Primer composition for amplifying a gene region having diverse variations in a target gene

A technology of primer composition and primer set, applied in the direction of combinatorial chemistry, organic compound library, recombinant DNA technology, etc., can solve the problems of RFMP analysis complexity, analysis difficulty, mass spectrum peak increase, etc.

Active Publication Date: 2015-04-15
GENEMATRIX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if several products different in nucleotide sequence from each other are amplified, there will be a problem that several different molecular weights are produced from one template
In addition, since RFMP is a method of analyzing genotypes by measuring the molecular weight of fragments generated from the amplification product of the gene region to be analyzed, if there are many molecular weight peaks in the mass spectrum, it makes RFMP analysis complicated and difficult
Therefore, if multiple primers are prepared based on the one-to-one correspondence of each genotype, and then mixed to amplify a gene region with multiple variations within the target gene and analyze the genotype of the gene region by "RFMP analysis", since Increased mass spectral peaks, with high potential to make their analysis more difficult and lead to erroneous results

Method used

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  • Primer composition for amplifying a gene region having diverse variations in a target gene
  • Primer composition for amplifying a gene region having diverse variations in a target gene
  • Primer composition for amplifying a gene region having diverse variations in a target gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1. HPV (Human Papillomavirus) Genotyping

[0095] Using the aforementioned restriction fragment mass polymorphism analysis (hereinafter, referred to as "RFMP analysis"), it was found that the genotype of human papillomavirus (hereinafter, referred to as "HPV") causes cervical cancer in humans. In this example, HPV DNA was analyzed.

[0096] HPV is a DNA virus belonging to the papovavirus virus family, which forms an icosahedron from 72 outer units (capsomers), and includes a double-stranded circular DNA composed of 7,900 nucleotides. Depending on the similarity of nucleotide sequences, HPV can be divided into 120 different subtypes. Thirty of these 120 subtypes are known to infect the lower genital tract. According to the risk of causing cervical intraepithelial neoplasia and cervical cancer, it is divided into high-risk types HPV-16, HPV-18, HPV-26, HPV-30, HPV-31, HPV-33, HPV-35, HPV- 39. HPV-45, HPV-51, HPV-52, HPV-53, HPV-56, HPV-57, HPV-58, HPV-59, HPV-6...

Embodiment 2

[0157] Example 2. HLA (Human Leukocyte Antigen)-DQB1 Genotyping

[0158] It is further confirmed whether the trigger primer (TP) and amplification primer (AP) of the present invention can be used to analyze various genotypes. For example, to genotype the HLA (human leukocyte antigen)-DQB 1 allele associated with the human immune response, RFMP analysis was performed using trigger primers (TP) and amplification primers (AP) after PCR amplification . Example 2 was tested with human genomic DNA.

[0159] 1. PCR amplification

[0160] A pair of amplification primers (AP) were prepared by aligning approximately 100 HLA-DQB1 sequences to determine "genotype-analyzable sequences" and selecting the binding portion of the primers. In addition, a trigger primer pair (TP) is prepared to complementarily bind to the specific sequences at the 3' and 5' ends of the HLA-DQB1*0201 type of the HLA-DQB1*0201 DNA template. PCR was performed using a mixture comprising the above-designed high c...

Embodiment 3

[0206] Example 3. Analysis of base variation of hepatitis B virus related to adefovir resistance

[0207] Occasional base mutations in the DNA polymerase gene of hepatitis B virus induce hepatitis B in humans. Through some mutations in bases, resistance to the drug Adefovir for the treatment of hepatitis B has emerged. Amino acid mutations in A181V / T and N236T have been reported to cause resistance to adefovir. It was further confirmed whether PCR amplification using the trigger primer (TP) and amplification primer (AP) of the present invention can be used to analyze various genotypes. In other words, the genotype of the adefovir-resistant hepatitis B virus was analyzed by RFMP analysis after PCR amplification using trigger primer (TP) and amplification primer (AP). Example 3 was performed on HBV DNA.

[0208] 1. PCR amplification

[0209] In order to analyze the nucleotide of the 236th amino acid of the DNA polymerase gene in the hepatitis B virus, the amplification prime...

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Abstract

The present invention provides a primer composition for amplifying a gene region having various variations in a target gene comprising: a first primer group comprising at least one primer specific to at least one template including a genotype-analyzable sequence of a genetically variant base sequence subjected to be analyzed in the target gene; and a second primer group comprising at least one primer designed by selecting one primer having the greatest number of shared bases from the primers of the first primer group and by using the selected primer as a reference, the primer of the second primer group binding specifically to the template or binding complementarily to the template with allowing mismatch of up to three successive bases or 1 to 2 bases in the vicinity of the 3' end of the template.

Description

technical field [0001] The present invention relates to a primer composition for amplifying a gene region that has multiple variations within a target gene, and also relates to a method for amplifying a target gene using the primer composition, comprising the primer A PCR amplification kit for the composition, and a method for genotyping a target gene, more particularly to a primer composition capable of simply and efficiently amplifying a gene region with multiple variations within the target gene, to complete without deletion Accurate amplification of a gene region, accurate analysis of a large number of genotypes while satisfying specificity and sensitivity conditions, a method for amplifying a target gene using the primer composition, a PCR amplification kit comprising the primer composition, and A method of genotyping a target gene using the primer composition. [0002] In addition, the present invention relates to a primer composition capable of producing an amplificati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q2600/156C12Q1/686C12Q1/708C12Q1/6876C12Q1/6881C12Q1/6872C12Q1/706C12Q1/6858C12Q2525/131C12Q2549/119C12Q2565/627C12Q2527/143C12Q2537/149C12Q1/683C40B40/06
Inventor 刘王敦黄善永申秀璟申智英李柱炯朴宣玟洪璿杓金锡俊金寿玉
Owner GENEMATRIX
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