• BMB Reports
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• 제38권5호
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• pp.555-562
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• 2005

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5% to 95% and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1% for both the approaches, allowing to extend the range of quantifications from 1% to 99%. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

• BMB Reports
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• 제29권1호
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• pp.45-51
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• 1996

Most expressed HLA loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino-terminal domain of the molecule. In this study, the HLA-DRB1 genotypes were determined in eighteen control cell lines and 112 unrelated Koreans using the PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primer) technique. 29 specific primer pairs in assigning the DRB1 gene were used. The results of control cells correlated well with the data which was previously reported. The heterozygosity and homozygosity of the DRB1 gene were 0.786 and 0.214, respectively. In a total of 41 different DRB1 alleles and 83 genotypes, the most frequent allele and genotype were DRB1*04 and DRB1*0901/1501, respectively. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-DRBI genotypes. Moreover, these results-allele and genotype frequency and heterozygosity of the HLA DRB1 gene-could be useful for database study before being applied to individual identification and transplantation immunity.

• BMB Reports
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• 제40권3호
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• pp.444-447
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• 2007

Polymerase chain reaction (PCR) is a powerful technique in molecular biology and is widely used in various fields. By amplifying DNA fragments, PCR has facilitated gene cloning procedures, as well as molecular genotyping. However, the extraction of DNA from samples often acts as a limiting step of these reactions. In particular, the extraction of PCR-compatible genomic DNA from higher plants requires complicated processes and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors, including polyphenolic compounds. We recently developed a novel solution, referred to as AnyDirect, which can amplify target DNA fragments directly from whole blood without the need for DNA extraction. Here, we developed a simple lysis system that could produce an appropriate template for direct PCR with AnyDirect PCR buffer, making possible the direct amplification of DNA fragments from plant leaves. Thus, our experimental procedure provides a simple, convenient, non-hazardous, inexpensive, and rapid process for the amplification of DNA from plant tissue.

• Molecules and Cells
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• 제24권1호
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• pp.60-68
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• 2007

Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The $(AG)_n$ motif was most common (23.1%), followed by the $(AAC)_n$ motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.

• 생명과학회지
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• 제15권1호
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• pp.45-48
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• 2005

서울대공원에서 사육중인 Miniature horse 10두의 혈통정립을 목적으로 PCR에 의한 성별 판정 및 16개 microsatellite marker를 사용하여 친자감정을 실시하였다. 성별 판정에서 430 bp의 SRY band가 관찰된 7두는 숫말로 판정되었고, 친자감정에서는 멘델의 유전양식에 부합된 3두가 친자관계가 확인되었다. 향후 Miniature horse의 혈통보존 및 관리에 유용한 자료가 될 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제18권6호
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• pp.762-766
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• 2005

This study was conducted to establish an individual identification system comprising of 19 microsatellite markers located on different bovine autosomes. The markers were typed on 257 animals from five cattle breeds. In total, 112 alleles were detected from the genotyping of 19 microsatellite markers. The average heterozygosities ranged from 0.292 to 0.824 and the polymorphic information content (PIC) ranged from 0.274 to 0.817 in Hanwoo. We found that there were differences in allele frequencies in Hanwoo when compared with other cattle breeds. The calculated cumulative power of discrimination (CPD) was 99.999% when nine microsatellite loci were used for analysis in the individual identification system. Also the matching probability, the probability that two unrelated animals would show the same genotypes, was estimated to be $0.44{\times}10^{-9}$. Therefore, the nine markers used in this study will be used for individual identification in two million Hanwoo individuals.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1515-1518
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• 2012

Aim: We conducted a prospective study in an Chinese population to detect the association between GSTM, GSTT and GSTP gene polymorphisms and survival of gastric cancer. Methods: A prospective follow-up study with 317 gastric cancer patients was conducted between January 2003 and January 2005. GSTM1, GSTT1 and GSTP1 genotyping was performed using ABI TaqMan Gene Expression assays. Results: Of 317 patients, 5 were lost to follow-up due to migration, while the remaining 302 patients completed the study. The median follow-up time was 34.2 months (range: 2 to 60 months), during which a total of 120 (39.1%) died of gastric cancer. The GSTT1-null genotype showed a significant increased risk of death from gastric cancer, with an HR (95% CI) of 1.59 (1.04-3.58). Moreover, we found individuals carrying null-GSTM1 and null-GSTT1 had a moderate higher risk of death from gastric cancer, with an HR of 1.92 (1.05-3.65). Conclusion: This study reported the carriage of null GSTT1 and null GSTM1 might be linked to the higher death risk from gastric cancer in Chinese population.

• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.6187-6190
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• 2012

We aimed to investigate DNA repair gene expression of response to chemotherapy among gastric patients, and roles in the prognosis of gastric cancer. A total of 209 gastric cancer patients were included in this study between January 2007 and December 2008, all treated with chemotherapy. Polymorphisms were detected by real time PCR with TaqMan probes, and genomic DNA was extracted from peripheral blood samples. The overall response rate was 61.2%. The median progression and overall survivals were 8.5 and 18.7 months, respectively. A significant increased treatment response was found among patients with XPG C/T+T/T or XRCC1 399G/A+A/A genotypes, with the OR (95% CI) of 2.14 (1.15-4.01) and 1.75 (1.04-3.35) respectively. We found XPG C/T+T/T and XRCC1 399 G/A+A/A were associated with a longer survival among gastric cancer patients when compared with their wide type genotypes, with HRs and 95% CIs of 0.49 (0.27-0.89) and 0.56 (0.29-0.98) respectively. Selecting specific chemotherapy based on pretreatment genotyping may be an innovative strategy for further studies.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7731-7735
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• 2014

Colorectal cancer (CRC) is the third most common cause of death due to cancer in the worldwide and the incidence is also increasing in Turkey. Our present aim was to investigate any association between germ-line methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and CRC risk in Turkey. A total of 86 CRC cases and 212 control individuals of the same ethnicity were included in the current study. Peripheral blood-DNA samples were used for genotyping by StripAssay technique, based on the reverse-hybridization principle and real-time PCR methods. Results were compared in Pearson Chi-square and multiple logistic regression models. The MTHFR 677TT (homozygous) genotype was found in 20.9% and the T allele frequency 4.2-fold increased in CRC when compared with the control group.The second SNP MTHFR 1298CC (homozygous) genotype was found in 14.0% and the C allele frequency 1.4-fold elevated in the CRC group. The current data suggest strong associations between both SNPs of germ-line MTHFR 677 C>T and 1298 A>C genotypes and CRC susceptibility in the Turkish population. Now the results need to be confirmed with a larger sample size.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7977-7980
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• 2014

This study was undertaken to evaluate the prevalence of significant cervical pathology among women who are high-risk human papillomavirus (HR-HPV)-positive/cytology negative, the most common combination of positive co-tests. The records of 244 women HR-HPV-positive/cytology-negative who had undergone colposcopy at Srinagarind Hospital, Khon Kaen University during January 2010 and April 2014 were reviewed. Mean age was 46.4 years. Of these 224 women, 75 were positive for HPV types 16/18 (33.5%) and 123 were positive for non-16/18 types (54.9%). HR-HPV was not genotyped in the remaining 26 women (11.6%). Prevalence of significant lesions for the entire cohort was 2.4%, and 2.6% and 3.3%, respectively, for those with HPV 16/18 and other oncogenic HPV types. One woman with HPV 16/18 (1.3%) had invasive cervical cancer. Multiparous women were more likely to be infected with HPV 16/18 compared to nulliparous women (36.3% versus 17.6%, respectively). In conclusion, the prevalence of significant cervical lesion among our study population was 2.4%. Multiparous women were more likely to be infected with HPV 16/18 compared to nulliparous women.