• The Journal of Dong Guk Oriental Medicine
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• v.7 no.2
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• pp.141-148
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• 1999

A simple and rapid FoLT(formamide low temperature)-PCR, whereby human genomic DNA from blood can be amplified without DNA preparative stps, is described using human insulin genes. By applicatin of FoLT-PCR in human insulin genes, intragenic polymorphism in non-coding regions of the human insulin gene was shown after amplification and analysis by restriction enzyme digestion. All nucleotide sequences were the same as the reported, and four necleotides, at 4 different positions were polymorphic, and polymorphic alleles ${\alpha}4$, ${\alpha}5$, ${\alpha}6$, and ${\beta}2$ were identified. The new alleles were originated from homologous recombination between the ${\alpha}1$ and ${\beta}1$ alleles, and the alleles were founded in heterozygotes only. Although allele ${\alpha}1$ was dominant, the new alleles and ${\beta}1$ were recessive. From the results, it was suggested that the new method of FoLT-PCR was highly applicable in genetic variation analysis.

• Journal of Plant Biotechnology
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• v.44 no.1
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• pp.89-96
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• 2017

The CRISPR/Cas9 is a core technology that can result in a paradigm for breeding new varieties. This study describes in detail the sgRNA design, vector construction, and the development of a transgenic plant and its molecular analysis, and demonstrates how gene editing technology through the CRISPR/Cas9 system can be applied easily and accurately. CRISPR/Cas9 facilitates targeted gene editing through RNA-guided DNA cleavage, followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. It also allows the generation of heritable-targeted gene mutations and corrections. Here, we present detailed procedures involved in the CRISPR/Cas9 system to acquire faster, easier and more cost-efficient gene edited transgenic rice. The protocol described here establishes the strategies and steps for the selection of targets, design of sgRNA, vector construction, and analysis of the transgenic lines. The same principles can be used to customize the versatile CRISPR/Cas9 system, for application to other plant species.

• Journal of Sasang Constitutional Medicine
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• v.9 no.2
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• pp.163-173
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• 1997

Amp-FLP is a one of the most frequently used human genetic analysis methods which adopts STR and VNTR typings. In this study, 100 genomic DNA samples of Taeum, Soyang and Soum constitution group were analysed by Amp-FLP method. The allele distribution of three STR loci(TH01, vWA and CSF1PO) and one VNTR locus(MCT118) of each different constitution group were investigated and the allele distribution was statistically evaluated. It was found out that the allele distribution of MCT118 and THO1 loci was not significantly different among different constitutions. However, the allele distribution of vWA showed p-value of 0.02056(Soyang group) and $2.41{\times}10^{-10}$(Soum group) which is much lower than significant level of p-value 0.05. Also, p-value of CSF1PO in Soum group was found out to be $4.65{\times}10^{-17}$. Therefore, it is expected that vWA and CSF1PO loci can be used as an indicator for gnenetic difference of different constiturion if the same result is obtained with sufficient number of samples.

• Korean Journal of Environmental Agriculture
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• v.37 no.2
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• pp.135-140
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• 2018

BACKGROUND: The Asian hornet (Vespa velutina nigrithorax), a wasp species, has attacked honey bee populations and affected the beekeeping industry in Korea over the past 15 years. However, little research has been done with this invasive species. In this study, we investigated the intestine bacterial microbiota of Asian hornets and honey bees to design an attractive trap for Asian hornets. METHODS AND RESULTS: Genomic DNAs isolated from the intestine microorganisms of Asian hornets and honey bees were utilized to amplify bacterial 16S rDNA for the comparative sequence analysis. The next generation sequencing analysis identified that the orders Flavobacteriales as the most abundant intestinal microorganisms in Asian hornets, showing a clear difference compared to honey bees in which Aeromonadales are dominant. We also report five newly identified 16S rDNA sequences of Asian hornet intestinal bacteria. According to the sequence blast search, these five bacteria belong to the genera Thalassomonas, Caedobacter, Vampirovibrio, Alkaliphilus and Calothrix. CONCLUSION: While Asian hornets and honey bees show similar intestine bacterial diversity, the relative ratio of bacterial populations is different. providing useful information to design pest control agents specifically targeting Asian hornets.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.33-40
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• 2010

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.116-121
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• 2010

Long-chain polyunsaturated fatty acids (LC-PUFA) promote the development of brain and vision of the fetus, relieve inflammation, inhibit oral dysplasia of rumor cell, decrease the incidence of cardiovascular disease and regulate arrhythmia. ${\Delta}-6$ desaturase is the rate-limited enzyme in the desaturation process. This study reports the cloning, characterization and tissue expression of a ${\Delta}-6$ desaturase gene in the chicken. PCR primers were designed based on the predicted sequence of chicken ${\Delta}-6$ desaturase (accession number: XM421053) and used to isolate a cDNA fragment of 1,323 bp from chicken liver. Based on the 1,323 bp fragment an EST (BI390105) was obtained by BLAST. The EST and 5'nd of the 1,323 bp fragment were partially overlapped. Gene specific primers derived from the EST were used for amplification of the 5'nd. Another gene-specific primer derived from the 1,323 bp fragment was used for amplification of the 3'nd by 3'ACE. Then the three overlapping cDNA sequences obtained were assembled with DNAMAN software and a full-length ${\Delta}-6$ desaturase of 2,153 bp was obtained. The full-length cDNA contained an ORF of 1,335 bp with a 5'ntranslated region of 147 nucleotides followed by an ATG initiation codon. Stop codon TGA was at position 1,481-1,483 bp. The deduced amino acids shared an homology above 77% with bovine, mice, orangutan, rat and human. The protein sequence had three histidine-rich regions HDFGH (HisI region), HFQHH (HisII region) and HH (HisIII region), a cytochrome $b_{5}$-like domain containing a heme-binding motif and two transmembrane domains. Sequence analysis of the chicken genomic DNA revealed that the coding sequence of chicken ${\Delta}-6$ desaturase included 12 exons and 11 introns. Semi-quantitative RT-PCR showed that the ${\Delta}-6$ desaturase expression levels were in turn liver, spleen, pancreas, lung, breast muscle, heart, and abdominal fat. The expression of ${\Delta}-6$ desaturase in liver was significantly higher than that in breast muscle (p<0.01). The expression of ${\Delta}-6$ desaturase in lung was significantly higher than that in abdominal fat (p<0.01). This is the first clone of chicken ${\Delta}-6$ desaturase.

• Journal of Korean Society of Forest Science
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• v.89 no.4
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• pp.536-542
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• 2000

A linkage map for Japanese red pine (Pinus densiflora) was constructed on the basis of two DNA marker systems of random amplified polymorphic DNAs (RAPDs) and inter-simple sequence repeats (I-SSR). Haploid genomic DNAs were extracted from megagametophyte tissues of 96 individual seeds in a single tree. A total of 98 DNA markers including 52 RAPD markers amplified by 25 primers and 46 I-SSR markers amplified by 18 primers were verified as Mendelian loci showing 1 : 1 segregation in 96 megagametophytes which were ${\chi}^2$-tested at 5% significance level. Of them, 63 segregating loci turned out to be linked into 20 linkage groups by the two-point analysis. However, 35 loci (17 RAPD and 18 I-SSR) of the 98 segregating loci did not coalesced into any linkage groups at a LOD of 3.0. The linked 63 loci were separated by an average distance of about 25.5 cM, which were spanned 1097.8 cM as a whole. The minimum and maximum map distances of the linkage groups were 4.3 cM and 54.9 cM, respectively. Incorporation of I-SSR loi into linkage map of RAPD loci resulted in extended and partially more saturated linkage blocks.

• Journal of Mushroom
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• v.13 no.1
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• pp.79-83
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• 2015

In this study, SCAR marker that differentiates Pleurotus eryngii strains with higher ${\beta}$-glucan from control strain was developed. Genomic DNAs of 9 control strains of Pleurotus eryngii and 9 Pleurotus eryngii strains with higher ${\beta}$-glucan were analyzed by bulked segregant analysis (BSA) using randomly amplified polymorphic DNA (RAPD). One-hundred twenty RAPD primers were screened on bulked DNA samples and a unique DNA fragment with the size of 91 bp was yielded by OP-R03 primer from the Pleurotus eryngii strains with higher ${\beta}$-glucan. A sequence characterized amplified region (SCAR) marker, designated as OP-R03-1-F and OP-R03-1-R, was designed on the basis of the determined sequence. The PCR analysis with the OP-R03-1 primer showed that this SCAR marker can clearly distinguish the Pleurotus eryngii strains with higher ${\beta}$-glucan from the control strains.

• Journal of Life Science
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• v.24 no.5
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• pp.522-531
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• 2014

Seasonal variations in the bacterial community of Gwangyang Bay seawater were analyzed using both isolation and cultivation-independent methods. Amplified rDNA restriction analysis was applied to 200 bacterial isolates. Bacterial isolates were composed of four phyla: Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Pyrosequencing was conducted, in addition to denaturing gradient gel electrophoresis (DGGE) of genomic DNA extracted directly from the water samples. The bacterial sequences obtained by pyrosequencing of 16S rRNA genes consisted of 24 phyla in the spring and summer, 39 in the fall, and 32 in the winter. The diversity index was high in the fall, whereas the dominancy index was high in the spring. In the spring, phylum Firmicutes was dominant, whereas phylum Proteobacteria dominated in the other three seasons. The second most dominant phyla were Proteobacteria in the spring, Firmicutes in the summer, and Bacteroidetes both in the fall and winter. Bacilliaceae was the most predominant family in the spring. Rhodobacteraceae and Bacilliaceae dominated in the summer, and Rhodobacteraceae dominated in the winter. Neither was dominant in the fall Twenty-seven bands purified from DGGE profiles were cloned and analyzed phylogenetically. In the spring, phylum Firmicutes dominated, followed by Proteobacteria. Proteobacteria dominated in all other seasons. Thus, two cultivation-independent methods for determination of seasonal variation patterns at the phylum level were in accordance with each other.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.5
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• pp.1426-1436
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• 2004

There have been several reports on the relationship between G protein β3 subunit gene (GNB3), angiotensin converting enzyme gene (ACE), β3-adrenergic receptor gene (ADRB3), and β2-adrenergic receptor gene (ADRB2) genotype and obesity or obesity related disease. The objective of this study was to examine the relationship between the combinations of these four genes' polymorphism and probability of obesity related disease in Korean female subjects. The experimental group was consisted of 85 obese Korean female subjects (body mass index, BMI≥27㎏/㎡). To determine the polymorphism, genomic DNA was isolated, and PCR was performed. Serological examinations (fasting plasma glucose, FPG; aspartate aminotranferase, AST; alanine aminotransferase, ALT; total cholesterol, TC; triglyceride, TG; high density lipoprotein-cholesterol, HDL; low density lipoprotein-choles terol, LDL) were carried by an autoanalyzer and serological methods. BMI, waist circumference (WC), hip circumference and waist hip ratio (WHR) were measured. Consequencely in the analysis with grouping of general genotyping and variant allele carrier/non-carrier, the result was not significantly different within all gene combinations and polymorphic pairings except higher waist circumference in Arg16Arg group of ADRB2 codon16 (P=0.024). And there was no significantly contrast result about age, height, weight, AST and ALT that are index feature of liver and gall bladder disease in polymorphic pairings of gene combinations. However, the statistical analysis of waist-hip ratio and waist circumference that could be recognized as the physical type of obesity showed T-Arg16 pairing carrier in GNB3-ADRB2 codon16 combination had increased WHR and WC significantly (P=0.046 and P=0.015 respectively). Futhermore, the levels of total cholesterol (TC) and low density lipoprotein choresteral (LDL) were significantly lower in C-I pairing of GNB3-ACE combination (P=0.032 and P=0.005). These results suggest that the T-Arg16 pairing carrier in GNB3-ADRB2 codon16 gene might have increased waist circumference and C-I pairing carrier in GNB3-ACE combination have lower possibility of contraction of cardiovascular disease related cholesterol and LDL despite of obese state.