DNA Microarray Probe Preparation by Gel Isolation Nested PCR

( Hong Min Wang ),( Wen Li Ma ),( Hai Huang ),( Wei Wei Xiao ),( Yan Wang ),( Wen Ling Zheng ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.3

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

DNA Microarray Probe Preparation by Gel Isolation Nested PCR

Wang, Hong-Min,Ma, Wen-li,Huang, Hai,Xiao, Wei-Wei,Wang, Yan,Zheng, Wen-Ling Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.3

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

방선균 유래 이차대사 생합성 유전자 분석용 DNA Microarray 제작 및 해석

남수정,강대경,이기형,김종희,강상순,장용근,홍순광,Nam Soo Jung,Kang Dae-Kyung,Rhee Ki Hyeong,Kim Jong-Hee,Kang Sang Sun,Chang Yong Keun,Hong Soon-Kwang 한국미생물학회 2005 미생물학회지 Vol.41 No.2

다양한 균주들을 대상으로 무작위로 신물질을 스크리닝하는 방법은 많은 노력과 시간이 소요되는 방법이며, 신물질을 발견하는 비율도 계속 낮아지고 있다. 따라서 기존 균주들을 대상으로 microarray 기술을 이용한 target-directed screening기술의 개발은, 학문적 뿐만 아니라 산업적으로도 중요한 의미를 가진다. 본 연구에서는, 이미 분리된 방선균각각의 유전체를 대상으로 microarray 분석을 통해, 새로운 생리활성 물질 생산균주 및 생합성 유전자를 확보할 수 있는 기법을 개발하기 위한 기초실험을 수행하였다. 즉, 기존에 알려진 생리활성물질 생합성 유전자들을 확보하여 DNA chip을 제조하였으며, 유전체 염기서열이 밝혀진 S. coelicolor 균주를 대상으로 그 효율성을 검증하였다. 전체적으로 유전자 상동성이 높을수록 반응감도도 높은 편이었으나, 이러한 상환관계가 일치하지 않는 유전자들도 있었다. 이와 같은 문제는, probe 유전자의 G+C 비율$(\%)$을 서로 비슷하게 구성하거나, 반응조건을 최적화 시킨다면 DNA chip의 효율성을 더욱 높일 수 있을 것으로 판단된다. DNA microarray를 통한 생리활성물질 발굴 연구는 세계적으로도 보고된 바 없는 새로운 접근방법으로서, 본 연구에서 시도하고 있는 방법은 발굴 target과 대상을 지정하고 시도되기 때물에, 효율면에서 무작위 스크리닝과는 비교되지 않을 정도로 높을것으로 예상된다. 또한 본 연구와 같은 접근방법을 최적화 시킨다면, 방선균뿐만 아니라 다른 미생물부터 생리활성물질 및 생합성유전자 스크리닝에도 효과적으로 응용할 수 있을 것이다. Streptomyces produces many kinds of secondary-metabolites including antibiotics. Screening of a new compound and elucidation of a biosynthetic pathway for the secondary metabolites are very important fields of biology, however, there is a main problem that most of the identified compounds are already researched compounds. To solve these problems, a microarray system that is based on the data related to the biosynthetic genes for secondary-metabolites was designed. For the main contents of DNA microarray, the important genes for the bio-synthesis of aminoglycosides, polyenes group, enediyne group, alpha-glucosidase inhibitors, glycopeptide group, and orthosomycin group were chosen. A DNA microarray with 69 genes that were involved in the bio-synthesis for the antibiotics mentioned above was prepared. The usability of the DNA microarray was confirmed with the chromosomal DNA and total RNA extracted from S. coelicolor whose genomic sequence had already been reported.

DNA microarray를 이용한 항진균 활성세균 Bacillus lentimorbus WJ5의 유전자 발현 분석

이영근,김재성,장유신,조규성,장화형 한국미생물학회 2003 미생물학회지 Vol.39 No.3

여러 항진균 활성 관련 유전자들의 발현 수준을 동시에 연구하기 위하여 DNA microarray를 이용하여 유전자들의 발현 패턴을 비교 분석하였다. 본 연구에서는 항진균활성을 가지는Bacillus lentimorbus WJ5의 genomic DNA를 무작위 하게 제한효소로 절단하여 2,000개의 DNA단편을 microarray하였으며, 감마선($^{60}Co$)조사로 유도된 7종의 항진균 활성 결핍 돌연변이체와 발현양상을 정량적으로 비교하였다. Gene Cluster (Michael Risen, Stanford Uniy.)를 이용한 DNA microarray의 분석 결과, 총 408개의 DNA 단편이 발현되는 것을 확인할 수 있었으며, 이들 중 20개의 DNA단편이 항진균 활성 결핍 돌연변이체에서 발현이 억제되는 것으로 나타났다. 특히,pbuX (xanthine permease, K222), ywbA (phosphotransferase system enzyme II, K393), ptsG (PTS glucose specific enzyme II ABC component, K877), yufO (ABC transporter(ATP-binding protein), K1301), 그리고 ftsY (signal recognition particle (docking protein), K868)는 모든 돌연변이체에서 동시에 발현되는 down-regulation된 유전자들로서 물질 이동과 관련된 것으로 보고되어 있으며, 항진균 활성 관련 신호 및 물질의 이동에 관여할 것으로 사료되어진다. The simultaneous expression levels of antifungal activity related genes was analyzed by DNA microarray. We constructed DNA chips contained 2,000 randomly digested genome spots of the antifungal bacterium of Bacillus lentimorbus WJ5 and compared its quantitative aspect with 7 antifungal activity deficient mutants induced by gamma radiation ($^{60}Co$). From the analysis of microarray hybridization by the Gene Cluster (Michael Eisen, Stanford Univ.), totally 408 genes were expressed and 20 genes among them were significantly suppressed in mutants. pbuX (xanthine permease, K222), ywbA (phosphotransferase system enzyme II, K393), ptsG (PTS glucose specific enzyme II ABC component, K877), yufO (ABC transporter (ATP-binding protein), K130l), and ftsY (signal recognition particle (docking protein), K868) were simultaneously down-regulated in all mutants. It suggested that they were supposed to be related to the antifungal activity of B. lentimorbus WJ5.

성매개 감염 진단을 위한 DNA Microarray 기반 검사의 임상적 유용성 평가

박애자,김소영,서동희 대한진단검사의학회 2016 Laboratory Medicine Online Vol.6 No.3

Background: Many molecular diagnostic methods have been developed to detect sexually transmitted infections (STI). The STDetect Chip (LabGenomics, Korea) which is a DNA microarray-based tool, newly developed for STI diagnosis in vitro, and the real-time PCR-based Anyplex STI-7 (Seegene, Korea) in clinical use were evaluated using ATCC DNA and clinical samples to determine the clinical usefulness of the STDetect Chip. Methods: The two methods were compared for consistency, sensitivity, and specificity for 6 pathogens in 300 prospectively selected clinical samples. Analytical sensitivity for ATCC Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis and Trichomonas vaginalis DNA and the effect of mixing bacterial DNA were studied. Results: The consistency of the two methods for clinical samples was superior at more than 0.92 kappa value. The sensitivity and specificity of the STDetect Chip compared with Anyplex STI-7 were 90.5-98.8%, and 95.6-99.6%, respectively. With similar analytical performance for ATCC DNA, the STDetect Chip detected 10-5 ng/μL of N. gonorrhoeae, 10-4 ng/μL of C. trachomatis, 10-6 ng/μL of M. hominis, and 10-3 ng/μL of T. vaginalis. For the mixture of three bacterial DNAs, less sensitive detection level was observed for T. vaginalis. Conclusions: The STDetect Chip showed good agreement with the Anyplex STI-7 test and it is considered clinically useful for detecting sexually transmitted pathogens. 배경: 성매개 감염의 진단을 위해 여러 가지 분자진단법이 개발되어 사용되고 있다. DNA microarray 기반의 성매개 감염 진단용 체외진단 의료기기로 새로이 개발된 STDetect Chip과 이미 임상에서 사용하고 있는 실시간 중합효소연쇄반응을 이용한 Anyplex STI-7에 대해 표준균주 DNA와 임상검체를 이용하여 두 가지 검사법의 분석적 성능 및 STDetect Chip의 임상적 유용성을 평가하고자 하였다. 방법: 전향적으로 얻어진 임상 검체 300건을 대상으로 6가지 병원체에 대해 두 가지 검사법의 일치도 및 임상 민감도 및 특이도를 분석하였다. Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis 그리고 Trichomonas vaginalis 네 가지 표준 균주에 대해 분석적 민감도 및 균주가 혼합되었을 때의 검출 영향에 대해 조사하였다. 결과: 임상 검체에 대한 검사 결과 일치도는 0.92 kappa 이상으로 매우 우수하였다. Anyplex STI-7과 비교한 STDetect Chip의 검사 민감도는 90.5-98.8%, 특이도는 95.6-99.6%를 보였다. 표준균주 DNA에 대해 전반적으로 유사한 분석적 성능을 보였으며, STDetect Chip의 경우 N. gonorrhoeae는 10-5 ng/L, C. trachomatis는 10-4 ng/L, M. hominis는 10-6 ng/L, T. vaginalis는 10-3 ng/L까지 검출하였다. 세 균주가 혼합되었을 때에 T. vaginalis의 검출능력이 떨어졌다. 결론: STDetect Chip은 Anyplex STI-7과의 검사 일치도가 높아, 성매개 감염 병원체 진단에 대해서 임상적 유용성을 가지는 것으로 판단된다.

Development of a Microarrayer for DNA Chips

Kim Sang Bong,Jeong Nam Soo,Kim Suk Yeol,Lee Myung Suk The Korean Society of Fisheries and Aquatic Scienc 2002 Fisheries and Aquatic Sciences Vol.5 No.1

Microarrayer is used to make DNA chip and microarray that contain hundreds to thousands of immobilized DNA probes on surface of a microscope slide. This paper shows the develop-ment results for a printing type of microarrayer. It realizes a typical, low-cost and efficient microarrayer for generating low density micro array. The microarrayer is developed by using a prependicular type robot with three axes. It is composed of a computer-controlled three-axes robot and a pen tip assembly. The key component of the arrayer is the print-head containing the tips to immobilize cDNA, genomic DNA or similar biological material on glass surface. The robot is designed to automatically collect probes from two 96-well plates with up to 12 pens at the same time. To prove the performance of the developed microarrayer, we use the general water types of inks such as black, blue and red. The inks are distributed at proper positions of 96 well plates and the three color inks are immobilized on the slide glass under the operation procedure. As the result of the test, we can see that it has sufficient performance for the production of low integrated DNA chip consisted of 96 spots within $1cm^2$ area.

마이크로배열 DNA의 감도 최적화

이주운 忠州大學校 2010 한국교통대학교 논문집 Vol.45 No.-

As the microarray technology has played an important role in broad range of science, more researchers are now relying on commercially available microarray products not only because of cost and time efficiency but also the quality. In this research, we aim to compare commercially available microarray slides and chemically modified DNA probes along with variation of spotting solution in terms of array sensitivity. Four different types of microarray slides, two different functional group linkers modified for nucleic acid probe, and glycerol content in spotting solution were evaluated. Microarray slides from Nexterion® coated with epoxy show highest signal intensity regardless of linker functional group. Addition of glycerol in spotting solution also improves the signal strength in general. Although this study is limited to particular commercial slides and chemical information of linker group is not disclosed by vendors, the work successfully demonstrates that optimum arraying protocol can be achieved and any incompatibilities of certain combinations can be found and avoided for future microarray spotting.

시토신 탈메틸화 관련 NtROS2a 유전자 발현을 제어한 RNAi 식물의 DNA microarray 분석

최장선,이인혜,정유진,강권규 한국식물생명공학회 2016 JOURNAL OF PLANT BIOTECHNOLOGY Vol.43 No.2

To study the transcript levels of epigenetically regulated genes in tobacco, we have developed a transgenic line OX1 overexpressing NtROS2a gene encoding cytosine DNA demethylation and a RNAi plant line RNAi13. It has been reported that salt- and H2O2- stress tolerance of these transgenic lines are enhanced with various phenotypic characters (Lee et al. 2015). In this paper, we conducted microarray analysis with Agilent Tobacco 4 x 44K oligo chip by using overexpression line OX1, RNAi plant line RNAi 13, and wild type plant WT. Differentially expressed genes (DEGs) related to metabolism, nutrient supply, and various stressed were up-regulated by approximately 1.5- to 80- fold. DEGs related to co-enzymes, metabolism, and methylation functional genes were down-regulated by approximately 0.03- to 0.7- fold. qRT-PCR analysis showed that the transcript levels of several candidate genes in OX1 and RNAi lines were significantly (p < 0.05) higher than those in WT, such as genes encoding KH domain-containing protein, MADS-box protein, and Zinc phosphodiesterase ELAC protein. On the other hand, several genes such as those encoding pentatricopeptide (PPR) repeat-containing protein, histone deacetylase HDAC3 protein, and protein kinase were decreased by approximately 0.4- to 1.0- fold. This study showed that NtROS2a gene encoding DNA glycosylase related to demethylation could regulate adaptive response of tobacco at transcriptional level. 담배에서 후성유전관련 유전자의 발현연구를 위해 담배유래 시토신 DNA 탈메틸화 관련 NtROS2a 유전자를 과발현 및RNAi 식물체를 육성하였다. 이들 형질전환체들은 고염 및산화 스트레스하에서 내성이 증진되었으며, 다양한 표현형변이를 보였다(Lee et al. 2015). 본연구에서는 선발된 과발현 (OX1), RNAi 식물체(RNAi 13) 및 대조식물체(WT)를 이용하여 Agilent Tobacco 4 X 44K Oligo chip으로 microarray분석을 수행하였다. OX1과 RNAi13 계통을 이용하여 WT과 함께비교 분석한 결과, 대부분 세포 내 이온 수송, 영양 공급 등과같은 물질대사와 생물적 비생물적 스트레스 및 methylation과관련되어 영향을 주는 유전자들에서 up-regulation 되었고, 물질대사관련 유전자와 세포 내 기능유전자의 역할을 담당하는 조효소, 그리고 다양한 스트레스 및 메틸레이션 관련유전자군에서 또한 down-regulation되었다. 각각의 up-, downregulation된유전자들을 WT과 비교하여 qRT-PCR을 수행한결과, KH domain-containing protein, MADS-box protein 및 Zinc phosphodiesterase ELAC protein 유전자들에서 발현이 높게나타났으며, 반면에 pentatricopeptide (PPR) repeat-containing protein, histone deacetylase HDAC3 protein 및 protein kinase는0.4 ~ 1.0-fold 발현양이 감소되었다. 따라서 DNA glycosylase 를 암호화하는 NtROS2a 유전자는 demethylation과 관련되어담배 식물체에서 다양한 전사레벨을 조절하는 것으로 판단된다.

시토신 탈메틸화 관련 NtROS2a 유전자 발현을 제어한 RNAi 식물의 DNA microarray 분석

최장선,이인혜,정유진,강권규,Choi, Jang Sun,Lee, In Hye,Jung, Yu Jin,Kang, Kwon Kyoo 한국식물생명공학회 2016 식물생명공학회지 Vol.43 No.2

담배에서 후성유전관련 유전자의 발현연구를 위해 담배유래 시토신 DNA 탈메틸화 관련 NtROS2a 유전자를 과발현 및 RNAi 식물체를 육성하였다. 이들 형질전환체들은 고염 및 산화 스트레스하에서 내성이 증진되었으며, 다양한 표현형변이를 보였다(Lee et al. 2015). 본연구에서는 선발된 과발현 (OX1), RNAi 식물체(RNAi 13) 및 대조식물체(WT)를 이용하여 Agilent Tobacco 4 X 44K Oligo chip으로 microarray분석을 수행하였다. OX1과 RNAi13 계통을 이용하여 WT과 함께 비교 분석한 결과, 대부분 세포 내 이온 수송, 영양 공급 등과 같은 물질대사와 생물적 비생물적 스트레스 및 methylation과 관련되어 영향을 주는 유전자들에서 up-regulation 되었고, 물질대사관련 유전자와 세포 내 기능유전자의 역할을 담당하는 조효소, 그리고 다양한 스트레스 및 메틸레이션 관련 유전자군에서 또한 down-regulation되었다. 각각의 up-, down-regulation된 유전자들을 WT과 비교하여 qRT-PCR을 수행한 결과, KH domain-containing protein, MADS-box protein 및 Zinc phosphodiesterase ELAC protein 유전자들에서 발현이 높게 나타났으며, 반면에 pentatricopeptide (PPR) repeat-containing protein, histone deacetylase HDAC3 protein 및 protein kinase는 0.4 ~ 1.0-fold 발현양이 감소되었다. 따라서 DNA glycosylase를 암호화하는 NtROS2a 유전자는 demethylation과 관련되어 담배 식물체에서 다양한 전사레벨을 조절하는 것으로 판단된다. To study the transcript levels of epigenetically regulated genes in tobacco, we have developed a transgenic line OX1 overexpressing NtROS2a gene encoding cytosine DNA demethylation and a RNAi plant line RNAi13. It has been reported that salt- and $H_2O_2$-stress tolerance of these transgenic lines are enhanced with various phenotypic characters (Lee et al. 2015). In this paper, we conducted microarray analysis with Agilent Tobacco 4 x 44K oligo chip by using overexpression line OX1, RNAi plant line RNAi 13, and wild type plant WT. Differentially expressed genes (DEGs) related to metabolism, nutrient supply, and various stressed were up-regulated by approximately 1.5- to 80- fold. DEGs related to co-enzymes, metabolism, and methylation functional genes were down-regulated by approximately 0.03- to 0.7- fold. qRT-PCR analysis showed that the transcript levels of several candidate genes in OX1 and RNAi lines were significantly (p < 0.05) higher than those in WT, such as genes encoding KH domain-containing protein, MADS-box protein, and Zinc phosphodiesterase ELAC protein. On the other hand, several genes such as those encoding pentatricopeptide (PPR) repeat-containing protein, histone deacetylase HDAC3 protein, and protein kinase were decreased by approximately 0.4- to 1.0- fold. This study showed that NtROS2a gene encoding DNA glycosylase related to demethylation could regulate adaptive response of tobacco at transcriptional level.

Improvement of Position of Label (POL) Influence by Fluorescently-labeled Helper Probe

윤현규,정인혁,오문주,김승준,황승용 한국바이오칩학회 2008 BioChip Journal Vol.2 No.3

DNA microarray-based technologies provide for high-throughput methods and the simultaneous analysis of a large number of genetic information at one assay. However, several important aspects of microarray technology such as the selection of optimal oligonucleotide probes and improvement of hybridization kinetics still have to be resolved. Recently, Lei Zhang et al. proposed that one of the important things in applications of a short oligonucleotide microarray is the position of label (POL) effect. In this study, we fabricated a microarray for the identification of polymorphisms in human mitochondrial DNA hypervariable region II (HVII), as well as a fluorescently-labeled helper probe applied to a fabricated microarray. In conclusion, we report on the results obtained with additional fluorescently-labeled oligonucleotide probes, called helper probes, in an attempt to improve the relative POL influences. Microarray results were improved and reduced the POL effect. These results may contribute to better hybridization patterns, data interpretation, and a new aspect for the selection of an efficient probe in microarray applications.