• BMB Reports
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• v.32 no.6
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• pp.529-534
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• 1999

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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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.

• Korean Journal of Medicinal Crop Science
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• v.18 no.4
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• pp.266-272
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• 2010

There is a considerable difference in morphological traits between Bokbunja cultivated in Korea (KCB) and Korea native Rubus coreanus, contrary to the conviction that the cultivated Bokbunja is the domestication of R. coreanus. To infer the phylogenetic relationship of KCB with other Rubus species, we compared the chloroplast DNA spacers of KCB with those of several Rubus species including black raspberry, R. occidentalis. The three chloroplast DNA spacers, atpB~rbcL, trnL~trnF, and trnT~trnL, were amplified using the specific primer pairs and converted to Single Strand Conformational Polymorphism (SSCP) markers. The SSCP makers of the chloroplast DNA spacers showed a considerable variation both within and among Rubus species. In the phylogenetic tree generated by the SSCP markers, KCB accessions were located in the same clade with R. occidentalis, but R. coreanus accessions in the different clade. Also, in the phylogenetic tree by the nucleotide sequences of the chloroplast DNA spacer trnL~trnF, KCB located in the same clade with R. occidentalis but not with R. coreanus. These results suggest that the three KCB accessions share higher similarity with R. occidentalis than with R. coreanus in the three chloroplast DNA spacers.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.7
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• pp.938-943
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• 2002

This study was perfonned to detect polymorphisms of the two candidate genes, bovine BTN (Butyrophilin) and ST AT5a (Signal Transducers and Activators of Transcription) gene using 98 Holstein bulls' frozen semen, and to offer the basic information for QTL (Quantitative Trait Loci) analysis. Each BTN PCR product was digested with endonuclease restriction enzyme. The digested fragments of four BTN PCR products were observed as follows: 316,280, and 162 bp in BTN1, 568, 305 and 263 bp in BTN2, 576, 332, and 244 bp in BTN3, and 573, 291, and 282 bp in BTN4, respectively. The gene frequencies of A and B allele in four BTN loci were as follows: 0.8980 and 0.1020 in BTN1, 0.5510 and 0.4490 in BTN2, 0.8163 and 0.1837 in BTN3, and 0.8875 and 0.1122 in BTN4, respectively. And three genotypes (homotypel, heterotype, and homotype2) for STAT5a were observed by SSCP (single stranded conformational polymorphism) method and the genotype frequencies are 78.57%, 19.39%, and 2.04%, respectively. The PlC (Polymorphism Information Content) value and heterozygosity of four BTN loci were as follows: 0.1695 and 0.1870 in BTN1, 0.3713 and 0.4927 in BTN2, 0.2549 and 0.2999 in BTN3, and 0.1794 and 0.1992 in BTN4, respectively. Comparing with the reported data, PlC value of BTN2 might have the possibility to be useful marker. Other BTN loci indicated skewed allele distribution.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.2
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• pp.165-169
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• 2005

This study was performed to offer effective basic data for selection and improvement of Korean dairy cattle through identifying distributional properties among candidate genes (bovine butyrophilin, signal transducers and activators of transcription 5a, and prolactin hormone). In this study, polymorphisms of candidate genes were identified and the relationships between loci and production traits of each gene were analyzed using frozen semen of Holstein bulls (19 proven and 77 candidates). In butyrophilin (BTN) locus, polymorphisms information contents (PIC) value of BTN2 (0.372) was higher than those of others (BTN1; 0.155, BTN3; 0.254, BTN4; 0.169). As a result of analysis of genotyping STAT5a, using single stranded conformational polymorphism (SSCP) method and microsatellite locus, PIC values were 0.189 and 0.457, respectively. And PIC value of prolactin hormone gene was 0.176. In the relationships between genotypes and production traits, BTN3 was associated with 305-day production traits (p<0.05). PTAs for B allele were such as 110.43, 88.28 and 75.25 in BTN1, 3, 4 and these values were higher than those of A allele, but in the case of BTN2, A allele with 154.19 was higher than that of B allele. The results obtained from using candidate genes may be used as an useful index for the genetic improvement of dairy cattle population in Korea, and further studies are needed.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.3309-3314
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• 2013

Spleen tyrosine kinase (SYK) is a non-receptor type cytoplasmic protein and a known tumor suppressor gene in breast cancer. Polymorphisms in SYK have been reported to be associated with cell invasion/cell morality and an increased risk of cancer development. In this case control study, all exons of the SYK gene and its exon/ intron boundaries were amplified in 200 breast cancer cases and 100 matched controls and then analyzed by single stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analyzed. Twelve variations were identified in exonic and intronic regions of DNA encoding SH2 domain and kinase domain of the SYK gene. All of these mutations are novel. Among them, 5 missense mutations were observed in exon 15 while one missense mutation was found in exon 8. In addition to these mutations, six mutations were also identified in intronic regions. We found a significant association between SYK mutations and breast cancer and observed that Glu241Arg, a missense mutation is associated with an increase risk of ~7 fold (OR=6.7, 95% CI=1.54-28.8), Thr581Pro (missense mutation) is associated with increased risk of ~16 fold (OR=15.5, 95%CI=2.07-115.45) and 63367 T>G (missense mutation) is associated with increased risk of ~13 fold (OR=12.8, 95%CI=1.71-96.71) for breast cancer. Significant associations were observed for each of these variations with both late menopause (p<0.01) and early menarche (p<0.005) cases when compared to controls. Our findings suggest that the polymorphic gene SYK may contribute to the development of breast cancer in at least the Pakistani population. This study provides an insight view of SYK which may provide a significant finding for the pharmaceutical and biotechnology industry.