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.
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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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