혼합 혈액 DNA 및 손상된 DNA시료에서 ABO 혈액형 유전자형 분석

엄기윤(Eom, Ki Yoon),조윤정(Cho, Yoon Jung),김효숙(Kim, Hyo Sook),문서현(Moon, Seo Hyun),황인관(?Hwang, In Kwan),강필원(Kang, Pil Won),김응수(Kim, Eung Soo) 경찰대학 경찰학연구편집위원회 2018 경찰학연구 Vol.18 No.3

본 실험은 9번 염색체 Exon 6, 7의 SNP를 target으로 하여 ABO genotyping을 수행하여 ABO 혈액형을 판정하는 실험이다. ABO genotyping 결과, AA 유전자형과 BB 유전자형 혈액 조합에서 AB 유전자형이, AA 유전자형과 OO 유전자형 조합에서는 AO 유전자형이 그리고 BB 유전자형과 OO 유전자형의 조합에서는 BO 유전자형과 유사한 결과를 나타낸다. 따라서, 실제 현장에서 이와 같은 오류를 나타낼 수 있는 두 가지 유전자형의 DNA를 다양한 농도 (10:1, 5:1, 1:1, 1:5, 1:10)로 혼합하여 ABO genotyping을 진행하였으며, DNA 혼합 비율에 따라 peak의 높이에 차이가 있음을 확인 할 수 있었다. 이러한 차이가 STR 분석에서도 확인되는지 시험하여 보았다. 그 결과 1:1 비율의 경우 완벽한 혼합시료임이 판별 되었고, 10:1, 5:1, 1:5, 1:10 비율의 경우 고농도 DNA의 STR 좌위가 우세하게 나타남을 확인하였다. 특히 10:1, 1:10 비율의 경우 drop out에 의해 한사람의 STR profile로 해석할 수 있는 가능성이 있었다. 따라서 STR 분석을 마친 DNA를 이용하여 ABO genotyping을 수행 할 경우, 한 사람의 STR profile로 판단된 경우일 지라도 사건 정황, DNA 혼합 여부 등을 고려하여 혈액형을 판정해야 한다. 또한, Degradation Index (DI) 값에 따른 DNA를 이용하여 ABO genotyping을수행한 결과 분해 상태가 심할수록 결과가 나빠지는 것을 확인했으며, 이를 통해 DNA 손상정도 역시 ABO genotyping 해석에 영향을 주는 주요한 요소 중 한 가지 임을 확인하였다. In this experiment, we performed ABO genotyping with the SNP of exon 6 and 7 on chromosome 9 to determine ABO blood type. In ABO genotyping, AB genotype is similar to the AA genotype mixed with the BB genotype, AO genotype is similar to the AA genotype mixed with the OO genotype and BO genotype is similar to the BB genotype mixed with the OO genotype. Therefore, ABO genotyping was performed by mixing two types of genomic DNA at different concentrations (10:1, 5:1, 1:1, 1:5, 1:10). It was confirmed that there is a difference in peak height depending on the DNA mixing ratio. We tested whether this difference was confirmed in the STR analysis as well. As a result, 1:1 ratio was confirmed to be a perfect mixed sample, and 1:5 ratio and 1:10 ratio, STR alleles of highly mixed DNA was predominant. Especially in case of 1:10 ration, STR profile could be a single source DNA by drop out. Therefore, when performing ABO genotyping using STR-finished DNA, blood type should be determined considering the case situation and DNA blending even if it is judged as one STR profile. In addition, ABO genotyping using DNA according to the Degradation Index (DI) value showed that the more the degradation condition, the worse the result, and the degree of DNA damage was also a major factor affecting the analysis of ABO genotyping

Distribution of HCV Genotypes in Chronic Korean HCV Patients

Lee, Kyung-Ok,Jeong, Su-Jin,Byun, Ji-Young,Shim, Ae-Sug,Seong, Hye-Soon,Kim, Kyung-Tae Korean Society for Clinical Laboratory Science 2007 대한임상검사과학회지(KJCLS) Vol.39 No.1

HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5'UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5'UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes. HCV는 single stranded RNA 바이러스로서 감염 시에는 만성간염 및 간경화 간암으로 진행될 수 있는 가능성이 높다. HCV는 6종의 주된 genotype과 그에 따른 많은 종류의 subtype이 보고되고 있으며, 세계 각 지역별로 그 분포는 매우 다양하다. 여러 가지 HCV genotype 중에서 1b 형에 감염되었을 경우 간경화나 간암으로 진행할 가능성이 높으며 치료효과도 떨어진다는 보고가 있어, 최근 HCV 환자의 치료에 있어서 HCV 바이러스 정량검사와 함께 HCV genotyping 검사의 임상적 활용이 높아지고 있다. 본 연구에서는 PCR-direct sequencing을 이용한 HCV genotyping 검사방법을 이용하여, 한국인 만성 HCV 간염환자에서 HCV genotype의 분포를 조사하였다. 검체로는 232명의 한국인 만성간염환자의 혈청을 사용하였으며, HCV 5'UTR 영역에서 선택한 2쌍의 primer로 nested PCR을 실시하였다. 증폭된 PCR산물 (215 bps)은 2% agrose gel로 전기영동을 하고 sequencing을 실시한 후 GeneBank의 BLAST 프로그램을 사용하여 HCV genotype을 분석하였다. HCV genotyping을 실시한 232명에서 5종류의 genotype, HCV 1b, 2a, 2b, 2c, 3a, 이 발견되었으며, HCV genotype 4, 5, 6 은 검출되지 않았다. 발견된 HCV genotype 중에서 HCV 1b의 검출률이 53.9%로 가장 높았고, 다음은 HCV 2a가 35.8%로 높게 나타나, 위 두 가지 HCV genotype을 합하면 거의 90%였다. 다음으로 HCV genotype 2b가 3.9%, 3a가 3.4% 그리고 2c가 3.0%의 순서로 검출되었다. 본 결과는 한국인 만성 HCV간염 환자의 치료 및 예후관리에 참고가 될 것으로 사료된다. 또한 PCR-direct sequencing을 이용한 HCV genotyping 검사는 간편하고 분명하게 결과를 판독할 수 있어 임상실험실에서 유용하게 사용될 수 있을 것으로 판단된다.

Distribution of HCV Genotypes in Chronic Korean HCV Patients

( Kyung Ok Lee ),( Su Jin Jeong ),( Ji Young Byun ),( Ae Sug Shim ),( Hye Soon Seong ),( Kyung Tae Kim ) 대한임상검사과학회 2007 대한임상검사과학회지(KJCLS) Vol.39 No.1

HCV는 single stranded RNA 바이러스로서 감염 시에는 만성간염 및 간경화 간암으로 진행될 수 있는 가능성이 높다. HCV는 6종의 주된 genotype과 그에 따른 많은 종류의 subtype이 보고되고 있으며, 세계 각 지역별로 그 분포는 매우 다양하다. 여러 가지 HCV genotype 중에서 1b 형에 감염되었을 경우 간경화나 간암으로 진행할 가능성이 높으며 치료효과도 떨어진다는 보고가 있어, 최근 HCV 환자의 치료에 있어서 HCV 바이러스 정량검사와 함께 HCV genotyping 검사의 임상적 활용이 높아지고 있다. 본 연구에서는 PCR-direct sequencing을 이용한 HCV genotyping 검사방법을 이용하여, 한국인 만성 HCV 간염환자에서 HCV genotype의 분포를 조사하였다. 검체로는 232명의 한국인 만성간염환자의 혈청을 사용하였으며, HCV 5``UTR 영역에서 선택한 2쌍의 primer로 nested PCR을 실시하였다. 증폭된 PCR산물 (215 bps)은 2% agrose gel로 전기영동을 하고 sequencing을 실시한 후 GeneBank의 BLAST 프로그램을 사용하여 HCV genotype을 분석하였다. HCV genotyping을 실시한 232명에서 5종류의 genotype, HCV 1b, 2a, 2b, 2c, 3a, 이 발견되었으며, HCV genotype 4, 5, 6 은 검출되지 않았다. 발견된 HCV genotype 중에서 HCV 1b의 검출률이 53.9%로 가장 높았고, 다음은 HCV 2a가 35.8%로 높게 나타나, 위 두 가지 HCV genotype을 합하면 거의 90%였다. 다음으로 HCV genotype 2b가 3.9%, 3a가 3.4% 그리고 2c가 3.0%의 순서로 검출되었다. 본 결과는 한국인 만성 HCV간염 환자의 치료 및 예후관리에 참고가 될 것으로 사료된다. 또한 PCR-direct sequencing을 이용한 HCV genotyping 검사는 간편하고 분명하게 결과를 판독할 수 있어 임상실험실에서 유용하게 사용될 수 있을 것으로 판단된다. HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5’UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5’UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes.

High-Resolution Melting Analysis에 의한 Duffy 혈액형군 유전형 분석

임채승,김경희,정소영,김윤호,최재열,김일태 대한수혈학회 2013 大韓輸血學會誌 Vol.24 No.1

Background:Accurate typing of Duffy blood group is important because anti-Duffy antibodies cause hemolytic transfusion reaction and hemolytic disease of the newborn. The aim of this study was to evaluate a new genotyping method using high resolution melting (HRM) analysis, a rapid and inexpensive approach for high-throughput Duffy genotyping. Methods:A total of 20 unrelated Korean blood samples were obtained and an African-black sample was used for GATA control. Phenotyping was performed by hemagglutination (DiaMed AG, Switzerland). GATA and FYA/B PCR products were obtained by PCR-restriction fragment length polymorphism (RFLP) using Taq DNA polymerase (Promega, WI) and enzymes BanI and StyI (New England Biolab, UK). For HRM, PCR amplification was performed using LightCycler 480 ResoLight Dye (Roche, USA) and Lightcycer 480 (Roche, USA). Results:Phenotyping and genotyping data using PCR-RFLP and HRM analysis were compared. Different types of HRM curves were obtained according to genotypes, FYA/FYA, FYB/FYB, and FYA/FYB, and to GATA mutations, homozygote FYB-33T (T/T), heterozygote FYB-33T/33C (T/C), and homozygote FYB-33C (C/C). Phenotypes 18 Fy(a+b−), 1 Fy(a+b+), 1 Fy(a−b+), and 1 Fy(a−b−) showed complete concordance with genotyping methods. Fy(a−b−) sample was found to be a FYB-33C homozygote by both genotyping methods. Conclusion:Phenotyping and genotyping showed concordant results and both genotyping methods using PCR-RFLP and HRM analysis showed good agreement in finding mutation in GATA and FY gene coding regions. HRM analysis is suitable and reliable for high-throughput screening for Duffy genotyping. 배경: Duffy 혈액형 항원에 대한 항체는 용혈성수혈부작용과 신생아용혈성질환을 일으키므로정확한 Duffy 혈액형 검사가 중요하다. 저자들은빠르고 경제적인 high-throughput이 가능한 새로운 high resolution melting (HRM) 분석법에 의한Duffy 유전형 검사를 평가하고자 하였다. 방법: 20개의 한국인 혈액 검체와 GATA 대조로써 흑인 검체 한 개에 대하여 다이아메드사의적혈구응집검사로 표현형을 확인하였고, Taq DNA polymerase와 BanI, StyI 효소를 사용하여 중합효소연쇄반응-제한절편길이다형성(PCR-RFLP) 검사를 시행하였다. HRM 분석을 위해 LightCycler 480 ResoLight Dye (Roche, USA)과 Lightcycer 480 (Roche, USA)으로 PCR 증폭을 시행하였다. 결과: 혈청학적 표현형과 PCR-RFLP와 HRM에의한 유전형 검사 결과를 비교하였다. HRM 분석결과, Duffy 유전형인 FYA/FYA, FYB/FYB, FYA/ FYB에 따라, 또한 GATA 유전자변이인 FYB-33T (T/T) 동형접합체, FYB-33T/33C (T/C) 이형접합체, FYB-33C (C/C) 동형접합체에 따라 HRM 곡선은 서로 다른 양상을 보였다. 표현형 검사 결과Fy(a+b−) 18건, Fy(a+b+) 1건, Fy(a−b+) 1건및 Fy(a−b−) 1건이 확인되었고 이는 두 가지 유전형 검사와 모두 일치하였다. Fy(a−b−) 검체는두 가지 유전형 검사법 모두에서 FYB-33C 동형접합체로 동일하였다. 결론: 혈청학적 표현형과 유전형 검사는 좋은일치율을 보였고 PCR-RFLP와 HRM에 의한 두가지 유전형 검사는 GATA와 Duffy 코딩부위 유전자 변이를 찾아내는 데에 동일한 결과를 보였다. HRM 분석법은 Duffy 유전자형의 high-throughput 선별검사로서 적합하며 신뢰할 수 있는 검사로 사료된다.

혈장 및 혈청 유리 핵산을 이용한 RhD 및 RhCEce 항원의 유전자형 분석

장호은,박경운,황상미,홍윤지,한민제,박정수,송정한,한규섭 대한수혈학회 2014 大韓輸血學會誌 Vol.25 No.3

Background:The Rh blood group includes several antigens, of which D, C, E, c, and e are clinically important. Although nucleic acids from whole blood can be used for Rh blood group genotyping, it is also possible to genotype free circulating fetal nucleic acids from plasma and serum. We performed Rh blood group phenotyping and genotyping using nucleic acids from whole blood and free circulating nucleic acids from plasma and serum, respectively. The results were compared. Methods:Forty-four blood samples were phenotyped and genotyped for RhD and RhCE blood groups. Phenotyping was performed by hemagglutination assay. Further tests were performed on RhD-negative samples. Nucleic acids were extracted from whole blood, plasma, and serum. Plasma and serum were prepared after filtration and genotyped by real-time polymerase chain reaction. Results:RhD blood group results showed one (2.3%) discrepant case in which the DEL phenotype appeared wild RHD genotype. Among nucleic acids, there were seven discrepant results: two from plasma and five from serum based on whole blood nucleic acids. RhCE blood group results showed three (6.8%) phenotype-genotype discordances. Among nucleic acids, seven (15.9%) cases were discrepant: one from plasma and six from serum compared to phenotypes. Kappa coefficients of serum were lower than those of plasma. Conclusion:RHD and RHCE genotype could be identified by assaying free circulating nucleic acids in plasma or serum. This study suggests that plasma is more reliable than serum as a specimen for RHD and RHCE genotyping of free circulating nucleic acids.

PCRㆍ직접염기서열분석법에 의한 ABO 유전형검사 6년 경험

원은정,조덕,허민석,박혜련,신명근,양동욱 대한수혈학회 2012 大韓輸血學會誌 Vol.23 No.3

Background: ABO genotyping is essential for resolving ABO grouping discrepancy and for determinating ABO subgroups. Most clinical samples, including suspected inherited subgroups and acquired variant phenotypes, can be determined by PCR-sequencing of exons 6 and 7 in the ABO gene. Here, we describe our six years' experience performing ABO genotyping by PCR-direct sequencing. Methods: We conducted a retrospective investigation of serological and genotypical data from 205 samples (158patients and 47 of their family members) of patients who were referred to the Molecular Genetics Laboratory at Chonnam National University Hwasun Hospital for ABO genotyping between January 2007 and July 2012. ABO genotyping was performed on all samples with PCR-direct sequencing of exons 6 and 7 in the ABO gene; the standard serologic tests were also performed. Results: The frequency of phenotypes consistent with their genotypes was 70.8% (112/158 cases) and the A2B3phenotype with the cis-AB01 allele was the most common (31.0%, 49 cases) among them. The frequency of phenotypes inconsistent with their genotypes was 29.1% (46/158 cases) and the A1B3 phenotype was the most frequently recovered case (5.1%, 8 cases). Family study showed differential phenotype expression depending on the co-inherited ABO allele in five families with the B306, cis-AB01, Ael02, Aw14, or B305 allele and also showed a typical inheritance of a chimera with A102/B101/O04. Conclusion: We propose that ABO genotyping using PCR-direct sequencing is useful for the resolution of ABO discrepancies and for the investigation of ABO subgroups based on six years' experience. In addition, family study for analysis of phenotypic patterns of ABO subgroups is also crucial to ABO genotyping. 배경: ABO 유전형 검사는 ABO 불일치의 해결및 ABO 아형의 결정에 있어서 매우 중요하다. 대부분 ABO 아형 및 변이 표현형을 보이는 임상검체에서 그 원인은 ABO 유전자의 엑손 6과 7의PCR증폭 및 직접염기서열분석을 통해 결정될 수있다. 본 저자들은 PCR증폭 및 직접염기서열분석을 통한 ABO 유전형 검사를 시행한 6년 간의경험을 소개하고자 한다. 방법: 본 연구는 2007년 1월부터 2012년 7월 사이에 화순전남대학교병원에 의뢰된 205 검체(환자 158명, 가족 47명의 검체)의 ABO 유전자검사결과를 후향적인 분석을 통하여 실시하였다. ABO 유전형 검사는 ABO 유전자의 엑손 6과 7에 대한PCR 증폭 후 직접염기서열분석을 통해 실시하였다. 표준혈청학적 검사 또한 시행되었다. 결과: ABO 유전형 검사가 의뢰된 총 158 검체중 112건(70.8%)은 ‘표현형과 유전형이 일치한 경우’였으며, cis-AB01 대립유전자를 동반하는 A2B3형이 49건(31.0%)으로 가장 흔했다. 표현형과 유전형이 일치하지 않은 경우는 158건 중 46건(29.1%)이었으며, 그들 중에서는 A1B3형이 8건(5.1%)으로 가장 높은 빈도를 보였다. B306, cis-AB01,Ael02, Aw14 그리고 B305 대립유전자를 가진 5발단자의 가계조사에서 동반되는 ABO 대립유전자에 따라 가족들이 다른 표현형을 보임을 보여주었고, A102/B101/O04를 갖는 키메라는 전형적인 키메라 유전양상을 보여주었다. 결론: 저자들은 ABO 유전형 검사를 시행한 6년 간의 경험을 통해 ABO 불일치의 해결과 ABO 아형의 감별에 있어서 PCR-직접염기서열분석법이 유용함을 밝히는 바이다. 또한 다양한 ABO 아형의 표현 양상을 분석할 수 있는 가계조사가 함께 이뤄져야 할 것이다.

대량 모근 시료 DNA 분리 체계 확립과 11 microsatellite maker를 사용하는 한우 생산이력제로의 적용가능성 검증

임현태,이상호,유채경,선두원,조인철,윤두학,양대용,정일정,이정규,전진태 경상대학교 농업생명과학연구원 2010 농업생명과학연구 Vol.44 No.6

한우 이력추적제에 적용되는 11개의 MS marker (TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, ETH3, ETH225, BM1824 and INRA23)와 성감별을 위한 2개의 sexing primer로 조합된 하나의 Multiplex PCR set를 이용하여 모근에서 추출한 genomic DNA를 이용해 3510두의 대량 시료를 분석한 결과 3.93%의 genotyping 실패율로 성공적인 분석결과를 얻었다. 무작위교배집단으로 가 정 시 동일개체출현확률 (PI)은 1.31×10-23, 반형매교배집단으로 가정 시 동일개체출현확률 (PIhalf-sibs)은 2.52×10-16 그리고 전형매교배집단으로 가정 시 동일개체출현확률 (PIsibs)은 1.09×10-6으로 나타나 현재 사용 중인 11종의 MS marker는 범용적으로 사용하여도 무방할 것으로 재확인 되었다. 또한 생산 및 사 육단계의 생우의 경우 모근을 이용하여 DNA를 추출하는 것은 시료 채취 시 소에게 주어지는 스트레스를 최소화 시킬 수 있을 뿐만 아니라 대립유전자형 분석에 있어서 시간적, 경제적인 효율성을 높일 수 있었 다. 또한 모근 채취 부위 중 등, 배, 꼬리상부와 꼬리하부를 이용하여 검정한 결과 꼬리하부의 모근을 이 용하여 5~13가닥을 사용했을 때 최적의 분석결과를 보였다. 최종적으로 한우의 사육단계 대량 유전자형 분석에 적용 가능한 96 well 단위를 기본으로 하는 모근 DNA분리 체계를 확립하였다. We used a multiplex PCR primer set composed of 11 microsatellite (MS) markers and two sexing markers for gender detection. Genomic DNA extracted from hair roots of 3,510 Hanwoo were genotyped. Based on the 11MS markers, no animals had identical genotypes(TGLA227, BM2113, TGLA53, ETF10, SPS115, TGLA122, ETH3, ETH225, BM1824 and INRA23). The expected probability of identity among genotypes of random individuals (PI), the probability of identity among genotypes from random half-sibs (PIhalf-sibs) and among genotypes of random individuals, and the probability of identity among genotypes from random sibs (PIsibs) were estimated as 1.31×10 -23 , 2.52×10 -16 and 1.09×10 -6 , respectively using the API-CALC program, version 1.0. We successfully completed the genotype analysis of 3,510 Hanwoo with a 3.93% genotyping failure rate. It was revealed that extracting DNA from the hair root was a time-efficient and cost-effective method to collect specimens for DNA isolation from live animals. This method also minimized stress for the animals during specimen collection. Among the hair roots from the back, belly, upper tail and lower tail, 5~13 hair roots of the lower tail led to the best genotype analysis results. Finally, we established a 96-well-format method of DNA preparation applicable for high- throughput genotype analysis.

Combinatorial approach to estimate copy number genotype using whole-exome sequencing data

Hwang, Mi Yeong,Moon, Sanghoon,Heo, Lyong,Kim, Young Jin,Oh, Ji Hee,Kim, Yeon-Jung,Kim, Yun Kyoung,Lee, Juyoung,Han, Bok-Ghee,Kim, Bong-Jo Elsevier 2015 Genomics Vol.105 No.3

<P><B>Abstract</B></P> <P>Copy number variations (CNVs) are known risk factors in complex diseases. Array-based approaches have been widely used to detect CNVs, but limitations of array-based CNV detection methods, such as noisy signal and low resolution, have hindered detection of small CNVs.</P> <P>Recently, the development of next-generation sequencing techniques has increased rapidly owing to declines in cost. Particularly, whole-exome sequencing has proved useful for finding causal genes and variants in complex diseases. Because gene copy number may affect expression, CNV genotyping can be very valuable in disease association studies. However, almost all current CNV detection tools consider only two types of CNV genotypes.</P> <P>In this study, we propose a CNV genotype estimation approach using a combination of existing methods. Our approach was comprehensively compared with the customized Agilent array–comparative genomic hybridization. We found that our genotyping approach proved to be accurate, and reproducible, suggesting that it can complement existing CNV genotyping methods.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CNV genotype estimation approach using a combination of existing methods </LI> <LI> Our approach was comprehensively compared with the customized Agilent array–comparative genomic hybridization. </LI> <LI> We found that our genotyping approach proved to be accurate, and reproducible, suggesting that it can complement existing CNV genotyping methods. </LI> </UL> </P>

High-risk Human Papillomavirus Genotype Detection by Electrochemical DNA Chip Method

Chansaenroj, Jira,Theamboonlers, Apiradee,Chinchai, Teeraporn,Junyangdikul, Pairoj,Swangvaree, Sukumarn,Karalak, Anant,Takahashi, Masayoshi,Nikaido, Masaru,Gemma, Nobuhiro,Poovorawan, Yong Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.4

High-risk human papillomavirus (HPV) genotypes are the major cause of cervical cancer. Hence, HPV genotype detection is a helpful preventive measure to combat cervical cancer. Recently, several HPV detection methods have been developed, each with different sensitivities and specificities. The objective of this study was to compare HPV high risk genotype detection by an electrochemical DNA chip system, a line probe assay (INNO-LiPA) and sequencing of the L1, E1 regions. A total of 361 cervical smears with different cytological findings were subjected to polymerase chain reaction-sequencing and electrochemical DNA chip assessment. Multiple infections were found in 21.9% (79/361) of the specimens, most prevalently in 20-29-year olds while the highest prevalence of HPV infection was found in the 30-39-year age group. The most prevalent genotype was HPV 16 at 28.2% (138/489) followed by HPV 52 at 9.6% (47/489), with the other types occurring at less than 9.0%. The electrochemical DNA chip results were compared with INNO-LiPA and sequencing (E1 and L1 regions) based on random selection of 273 specimens. The results obtained by the three methods were in agreement except for three cases. Direct sequencing detected only one predominant genotype including low risk HPV genotypes. INNO-LiPA identified multiple infections with various specific genotypes including some unclassified-risk genotypes. The electrochemical DNA chip was highly accurate, suitable for detection of single and multiple infections, allowed rapid detection, was less time-consuming and was easier to perform when compared with the other methods. It is concluded that for clinical and epidemiological studies, all genotyping methods are perfectly suitable and provide comparable results.

Genotyping-by-Sequencing: a Promising Tool for Plant Genetics Research and Breeding

정용석,김창수,최상철,전태환 한국원예학회 2017 Horticulture, Environment, and Biotechnology Vol.58 No.5

Plant genetics studies and breeding programs utilize the genetic variation caused by DNA polymorphisms. Molecular makers are used to detect these variations in the DNA. The advent of next-generation sequencing (NGS)technologies has conferred new opportunities for high-throughput genotyping in various plant species. Recent improvementsin high-throughput sequencing have enabled sequences to be used to detect and score single nucleotide polymorphisms(SNPs) by bypassing the time-consuming process of marker development. With NGS, whole-genome sequencing dataand millions of genome-wide SNPs for high-throughput genotyping have become available for a variety of geneticstudies and breeding programs. SNP markers are powerful for analyses of integrated SNP sets in a species, althoughhigh costs hinder the wider use of SNPs. However, genotype-by-sequencing (GBS), a series of genetic analyses thatincludes molecular marker discovery and genotyping using NGS technologies, has opened new possibilities in plantbreeding and plant genetics studies. It offers cost-effective genome-wide scanning and multiplexed sequencing platforms. The GBS method uses restriction enzymes coupled with DNA barcoded adapters to reduce complexity. GBS cansimultaneously perform SNP discovery and genotyping with or without reference genome sequences. Therefore, GBScan be applied to various approaches for plant breeding and plant genetics studies, including linkage maps, genome-wideassociation studies, genomic selection, and genomic diversity studies. These features make GBS an ideal tool for studiesranging from single-gene markers to whole-genome profiling.