• 한국미생물·생명공학회지
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• 제31권4호
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• pp.322-328
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• 2003

TAR(Transformation-Associated Recombination) cloning 법은 복잡한 고등생물의 게놈으로부터 유전자나 특정 염색체 부위를 선별적 분리를 가능하게 한다. 이 방법은 목적으로 하는 염색체 부위의 주변에 존재하는 비교적 짧은 게놈 염기서열에 대한 정보를 필요로 하며, spheroplast 형질전환 과정에서 게놈 DNA와 5'-와 3'-표적배열을 지닌 TAR vector 사이에서 일어나는 상동성 재조합과정에 의해 이루어진다. 본 연구에서는 single-copy 유전자의 클론닝에 필요한 specific hook의 최소 크기를 조사하였고, 서로 다른 TAR vector(radial과 unique vector)의 유용성을 조사하기 위해 동일한 single-copy 유전자의 클론닝을 통해 비교하였다. 그 결과, hHPRT 유전자에 대한 TAR cloning의 빈도는 hook의 길이가 750 bp∼63 bp의 범위에서 동일하게 나타났다. radial hook을 사용한 경우보다 unique hook을 사용하였을 경우 형질전환체의 수는 약 20배정도의 감소를 보였으나 목적으로 하는 재조합체의 분리 빈도는 두 배 이상 증가하였다. 그러므로 본 연구에서 two-unique TAR vector는 선별력이 높으므로 일반적 TAR cloning에 사용할 수 있으며, radial TAR vector의 경우는 병리학적 표본과 같이 제한된 게놈 DNA를 사용하는 경우에 더 적합하다고 볼 수 있다. 또한, single-copy 유전자의 분리에 필요로 하는 specific hoot의 최소 길이는 약 60 bp로도 가능하다는 것을 확인하였다.

• 대한유전성대사질환학회지
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• 제5권1호
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• pp.94-107
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• 2005

Prenatal diagnosis (PND) such as amniocentesis or chorionic villi sampling has been widely used in order to prevent the birth of babies with defects especially in families with single gene disorderor chromosomal abnormalities. Preimplantation genetic diagnosis (PGD) has already become an alternative to traditional PND. Indications for PGD have expanded beyond those practices in PND (chromosomal abnormalities, single gene defects), such as late-onset diseases with genetic predisposition, and HLA typing for stem cell transplantation to affected sibling. After in vitro fertilization, the biopsied blastomere from the embryo is analyzed for single gene defect or chromosomal abnormality. The unaffected embryos are selected for transfer to the uterine cavity. Therefore, PGD has an advantage over PND as it can avoid the risk of pregnancy termination. In this review, PGD will be introduced and application of PGD in inborn error metabolic disorder will be discussed.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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• pp.1119-1126
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• 2018

Objective: Present investigation was aimed to study the Single Nucleotide Variants of the luteinizing hormone beta ($LH{\beta}$) gene and to analyze their association with the semen quality (fresh and post-thawed frozen semen) and luteinizing hormone (LH) concentrations in Murrah buffalo bulls. Methods: Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and Sanger sequencing method is used to study genetic variability in $LH{\beta}$ gene. LH assay was carried out using enzyme-linked immunosorbent assay method. A fixed general linear model was used to analyze association of single nucleotide polymorphism (SNP) of $LH{\beta}$ gene with semen quality in 109 and LH concentrations in 80 Murrah bulls. Results: $LH{\beta}$ gene was found to be polymorphic. Total six SNPs were identified in $LH{\beta}$ gene g C356090A, g C356113T, g A356701G, g G355869A, g G356330C, and g G356606T. Single Stranded Conformational Polymorphism variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on sperm concentration (million/mL), percent mass motility, acrosome integrity and membrane integrity in fresh and frozen semen whereas significant (p<0.05) effect was observed on percent live spermatozoa. SSCP variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on luteinizing hormone concentrations too. Conclusion: The observed association between SSCP variants of $LH{\beta}$ gene with semen quality parameters and LH concentrations indicated the possibilities of using $LH{\beta}$ as a candidate gene for identification of markers for semen quality traits and LH concentrations in Murrah buffaloes.

• 한국축산식품학회지
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• 제32권6호
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• pp.776-782
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• 2012

Marbling (intramuscular fat) is the most economically important meat quality trait in Hanwoo (Korean cattle). The endothelial differentiation G-protein coupled receptor 1 (EDG1) gene, involved in blood vessel formation, is located within the genomic region of a quantitative trait locus (QTL) for marbling on bovine chromosome 3. Thus, the EDG1 gene can be considered as a positional and functional candidate gene for meat quality in beef cattle. This study aimed to identify single nucleotide polymorphisms (SNPs) in the EDG1 gene and to evaluate their associations with carcass traits in Hanwoo population. We have sequenced a fragment of 5'-UTR of the EDG1 gene and identified one SNP. Genotyping of the g.166A>G SNP marker was carried out using PCR-RFLP analysis in 309 Hanwoo steers in order to evaluate their association with carcass traits. The g.166A>G SNP marker showed a significant effect on the marbling score. Animals with the GG genotype had higher marbling score compared with AA and AG genotypes (p<0.05). This SNP marker also showed a significant additive effects for the marbling score (p<0.05). These results suggest that the EDG1 gene can be used as a molecular marker for DNA marker-assisted selection in order to increase the levels of the marbling score in Hanwoo.

• Korean Chemical Engineering Research
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• 제47권5호
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• pp.538-545
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• 2009

본 총설에서는 주사형프로브현미경의 원리와 응용에 관하여 간략히 설명하고 최근 본 그룹에 의하여 활발하게 연구되고 있는 나노탐침과 AFM(원자간력현미경 atomic force microscopy)을 이용한 저침습성(low-invasive) 단일세포 조작기술과 고효율 유전자 도입기술을 소개하고자 한다. 시판 AFM 탐침을 침상구조로 가공한 나노탐침과 AFM을 이용하였을 경우, 탐침의 세포삽입의 성공여부를 force-distance curve 상의 척력소실의 유무로 판단할 수 있다. 침상 나노탐침을 사용하면 대부분의 세포에서 80~90%의 고효율 세포삽입이 가능하여 마이크로인젝션용 미세관을 이용하는 경우보다 세포삽입효율이 높았다. 또한 나노탐침의 직경이 400 nm 이하의 경우에는 세포 종류에 관계없이 장시간 나노탐침의 삽입에도 세포활성에 큰 영향이 없었다. 침상나노탐침을 이용하여 DNA를 도입하였을 경우에도 기존의 DNA 도입방법과 비교하여 높은 도입효율과 유전자 발현율로 DNA를 도입할 수 있는 가능성을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5241-5243
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• 2012

Background: Prostate cancer (Pca) is one of the most common complex and polygenic diseases in men. The X-ray repair complementing group 1 gene (XRCC1) is an important candidate in the pathogenesis of Pca. The purpose of this study was to evaluate the association between single nucleotide polymorphisms in the XRCC1 gene and susceptibility to Pca. Materials and Methods: XRCC1 gene polymorphisms and associations with susceptibility to Pca were investigated in 193 prostate patients and 188 cancer-free Chinese men. Results: The c.910A>G variant in the exon9 of XRCC1 gene could be detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods. Significantly increased susceptibility to prostate cancer was noted in the homozygote comparison (GG versus AA: OR=2.95, 95% CI 1.46-5.42, ${\chi}^2$=12.36, P=0.001), heterozygote comparison (AG versus AA: OR=1.76, 95% CI 1.12-2.51, ${\chi}^2$=4.04, P=0.045), dominant model (GG/AG versus AA: OR=1.93, 95% CI 1.19-2.97, ${\chi}^2$=9.12, P=0.003), recessive model (GG versus AG+AA: OR=2.17, 95% CI 1.33-4.06, ${\chi}^2$=8.86, P=0.003) and with allele contrast (G versus A: OR=1.89, 95% CI 1.56-2.42, ${\chi}^2$=14.67, P<0.000). Conclusions: These findings suggest that the c.910A>G polymorphism of the XRCC1 gene is associated with susceptibility to Pca in Chinese men, the G-allele conferring higher risk.

• Asian-Australasian Journal of Animal Sciences
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• 제29권3호
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• pp.315-320
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• 2016

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

• 동의생리병리학회지
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• 제21권5호
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• pp.1307-1312
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• 2007

This study was designed to investigate effectiveness of korean medicine therapy and the relation between effectiveness of that and single nucleotide gene polymorphism in stroke patients. This study was carried out on 92 stroke patients who were admitted to the department of acupuncture & moxibustion, college of Oriental medicine, Daegu Haany University and 112 healthy Korean. All patients were received Korean medicine therapy including acupuncture and herbal medicine for stroke and assessed by National Institutes of Health Stroke Scale(NIHSS). Blood samples from all subjects were obtained for DNA extraction. The extracted DNA was amplified by polymerase chain reaction(PCR). PCR products were visualized by 1.5% agarose gel electrophoresis. Through Pyrosequencing of PCR product, the polymorphism of single nucleotide gene was genotyped automatically. There were significant difference between before and after Korean medicine therapy in NIHSS. Genotypes were AA, AG, GG, but there was no significant difference between control and stroke groups. And there was not any statistical significant allelic frequency difference between control and stroke groups. We concluded that Korean medicine therapy in stroke patient can improve NIHSS, but there is no definite relation between effectiveness of Korean medicine therapy and single nucleotide gene polymorphism in stroke patients. This study need to be confirmed in large patients and further studies about relation with gene polymorphism are required.

• 대한의생명과학회지
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• 제10권2호
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• pp.75-84
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• 2004

Massive identification of differentially expressed patterns has been used as a tool to detect genes that are involved in disease related process. We employed circular single stranded sense molecules as probe DNA for a DNA chip. The circular single stranded DNAs derived from 1,152 unigene cDNA clones were purified in a high throughput mode from the culture supernatant of bacterial transformants containing recombinant phagemids and arrayed onto silanized slide glasses. The DNA chip was examined for its utility in detection of differential expression profile by using cDNA hybridization. Hybridization of the single stranded probe DNA were performed with Cy3- or Cy5-labeled target cDNA preparations at $60^\circ$C. Dot scanning performed with the hybridized slide showed 29 up-regulated and 6 down-regulated genes in a cancerous liver tissue when compared to those of adjacent noncancerous liver tissue. These results indicate that the circular single stranded sense molecules can be employed as probe DNA of arrays in order to obtain a precious panel of differentially expressed genes.

• BMB Reports
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• 제28권6호
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• pp.484-489
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• 1995

The product of bacteriophage T7 gene 2.5 is a single-stranded DNA binding protein and plays an important role in T7 DNA replication, recombination, and repair. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth (Kim and Richardson, 1993). The C-terminal truncated gene 2.5 protein ($GP2.5-{\Delta}21C$) cannot substitute for wild-type gene 2.5 protein in vivo; suggesting that the C-terminal domain of gene 2.5 protein is essential for protein-protein interactions (Kim and Richardson, 1994; J. Biol. Chem. 269, 5070-5078). Truncated gene 2.5 proteins lacking 19 residues ($GP2.5-{\Delta}19N$) and 39 residues ($GP2.5-{\Delta}39N$) from the amino-terminal domain were constructed by in vitro mutagenesis. $GP2.5-{\Delta}19N$ can support the growth of T7 phage lacking gene 2.5 while $GP2.5-{\Delta}39N$ cannot substitute for wild-type gene 2.5 protein in vivo; however, its ability to bind to single-stranded DNA is not affected. These results clearly demonstrate that the 20~39 amino-terminal region of gene 2.5 protein is required for T7 growth in vivo but may not be involved in DNA binding activity.