Method of evaluating earwax type or underarm odor
An evaluation method, earwax technology, applied in the direction of botany equipment and method, biochemical equipment and method, microbial measurement/testing, etc., can solve the problem of not knowing the polymorphism of earwax type
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Embodiment 1
[0110] Example 1: Determination of earwax-determining genes
[0111] Known polymorphic markers in the about 5.9cM region around the cerumen locus described in previous studies by the present inventors are rare, so we firstly identified them in the NCBI Gene Bank (Genbank, http: / / www.ncbi.nih.gov / GenBank) for CA repeat markers. At the same time, in the JSNP database (http: / / snp.ims.u-tokyo.ac.jp), the single nucleotides related to the two genome sequences (AC003034 and AC007728) that should have been determined for this cerumen locus were retrieved acid polymorphism (SNP).
[0112] Corresponding to the genome sequence recorded in Genbank, 134 fluorescent dye marker primer sets were designed to amplify the CA repeat marker in the interval between AC003034 and AC007728 sequences. This interval contains the previously mapped D16S3039, D16S3117 and cerumen loci (Tomita et al., 2002).
[0113] Then, using these markers, the genotypes of 118 volunteers (64 dry-type individuals ...
Embodiment 2
[0131] Example 2: Frequency of Allele A and Alleles with 27 Base Deletions in Various Populations Rate distribution
[0132] So far, many studies have been carried out on the frequency of dryness-cerumen phenotype among various groups (Matsunaga, 1962; Petrakis et al., 1967; Petrakis, 1969; Martin and Jackson, 1969; Petrakis et al., 1971 ; Alfred et al., 1970; Omoto, 1973; Patel, 1973; Norakmal and Tan, 1979; Spitsyn and Afanasèva, 1989) (tables and online tables). For the genotypes of a total of 33 populations in the world (with one nationality or one ethnicity in one country as one population), the inventors performed direct sequencing, or, using the allele "A" specific The properties of Taqman probes were determined by real-time quantitative PCR or single-strand polymorphism analysis (SSCP) method.
[0133] As can be inferred from the phenotypic distribution of dry cerumen shown in previous studies, the frequency of allele A is extremely high in East Asians. The highe...
Embodiment 3
[0147] Example 3: Regarding the introduction of allele A or allele G of the human ABCC11 gene Assays for gene expression and cGMP transport in LLC-PK1 cells
[0148] Recombine the full-length cDNA with allele A or G of the human ABCC11 gene into the plasmid pcDNA3.1-Hygro, and use lipofection reagent (Invitrogen, Carlsbad, California) to transfect porcine-derived LLC- PK1 cells (JCRB cell bank). After 48 hours, the cells were transferred to culture medium containing 200 μg / ml hygromycin B. After 14 days, colonies of hygromycin-resistant cells were isolated. Total RNA was extracted from cells expressing allele A or allele G, and the expression level was identified by real-time quantitative PCR (RT-PCR) using 7900HT Sequence Detection System (Applied Biosystems). The ratio of ABCC11-mRNA and GAPDH (control) was calculated for statistical comparison. As a result, the copy number of each allele introduced was unknown, but the expression level of allele A was about 3 times h...
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