Genomic DNA: Purification

2018 ◽  
pp. 1-5 ◽  
Author(s):  
Burkhard Tümmler ◽  
Frauke Stanke
Keyword(s):  
Genomic Dna ◽  
Dna Purification

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

scholarly journals Optimasi Isolasi Genom untuk Analisis Keragaman Mikrob pada Fermentasi Singkong "Peyem" dengan Teknik Terminal Restriction Fragment Length Polymorphism (T-RFLP)

2013 ◽  
Vol 18 (1) ◽  
Author(s):  
Tati Barus
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Genomic Dna ◽  
Fragment Length Polymorphism ◽  
Dna Purification ◽  
Restriction Fragment Length

"Peyem" merupakan salah satu pangan fermentasi Indonesia. Kualitas pangan fermentasi bergantung pada aktivitas mikrob yang terdapat selama proses fermentasi berlangsung. Salah satu teknik molekuler yang telah banyak digunakan untuk menganalisis komunitas mikrob pada suatu habitat adalah teknik Terminal–Restriction Fragment Lenght Polymorphism (T-RFLP). Metode isolasi genom dan jenis primer yang digunakan pada saat PCR penting pada teknik T- RFLP dalam mengkaji komunitas mikrob. Oleh sebab itu, penelitian ini bertujuan untuk membandingkan empat metode isolasi genom dan membandingkan penggunaan dua set primer dalam mengkaji komunitas bakteri dari "Peyem" dengan teknik T-RFLP. Genom komunitas bakteri diisolasi dengan menggunakan empat metode, yaitu: 1) QIAamp DNA Stool Mini Kit (G1), 2) QIAamp DNA Stool Mini Kit + lisozim (G2), 3) Genomic DNA Purification Kit (G3), dan 4) Genomic DNA Purification Kit + lisozim (G4). Untuk mengamplifikasi 16S rDNA digunakan dua set primer, yaitu: 1) primer 27F-FAM dan 1492R, 2) primer 63F-FAM dan 1387R. Hasil penelitian menunjukkan isolasi genom dengan metode G4 menghasilkan konsentrasi genom tertinggi (330,20 ng/µl) dibandingkan metode G1, G2, dan G3 (163,50 ng/µl; 183,25 ng/µl, dan 260,80 ng/µl). Primer 27F-FAM menghasilkan jumlah peak yang lebih tertinggi (264) dibandingkan dengan primer 63F-FAM (177). Jumlah peak TRF pada teknik TRFLP menggambarkan keragaman komunitas mikrob. Dengan demikian isolasi genom dengan Genomic DNA Purification Kit + lysozyme dan penggunaan pasangan primer 27F-FAM-1492R adalah yang terbaik untuk menganalisis komunitas bakteri dari "Peyem" dengan teknik T-RFLP.Kata kunci: Genom, Primer, T-RFLP, Mikrob, "Peyem"

OCORRÊNCIA DE blaKPC E blaVIM EM ISOLADOS DE Pseudomonas aeruginosa MULTIDROGA RESISTENTES PROVENIENTES DE INFECÇÃO EM PACIENTES DE UM HOSPITAL PÚBLICO EM RECIFEPE.

2021 ◽  
Author(s):  
Érica Maria de Oliveira ◽  
Nathaly Bruna de Oliveira Silva ◽  
Ana Catarina de Souza Lopes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genomic Dna ◽  
Dna Purification

Introdução: Pseudomona aeruginosa é uma bactéria gram negativa oportunista considerada uma séria ameaça à saúde pública e são responsáveis por uma variedade de Infecções Relacionadas à Assistência à Saúde (IRAS), como pneumonias, infecções em tecidos moles, no trato urinário, e bacteremias(1).A emergência de P. aeruginosa resistentes aos carbapenêmicos, produtora de carbapenemases, dificulta ainda mais o tratamento e são associadas a altas taxas de mortalidade(2). Objetivos: O objetivo desse estudo foi analisar o perfil de susceptibilidade bem como detectar a presença dos genes de resistência para carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP e blaGES) em dois isolados de P. aeruginosa (Ps1-A2 e Ps2-A2) provenientes de secreção traqueal e ponta de cateter respectivamente, de dois pacientes de um hospital público de Recife-PE no ano de 2021. Métodos: A identificação bioquímica e o perfil de susceptibilidade dos isolados foi realizada através do sistema automatizado BD PhoenixTM. O DNA total foi extraído utilizando o kit comercial Wizard genomic DNA Purification e a pesquisa dos genes de resistência foram realizadas por Reação em Cadeia da Polimerase (PCR). Resultados: O isolado Ps1-A2 foi resistente a todos os antibióticos testados, com exceção da polimixina B. Já o isolado Ps1-A2 apresentou sensibilidade a ampicilina, gentamicina e polimixina B. Os genes blaNDM, blaIMP e blaGES não foram detectados. O gene blaKPC foi detectado no isolado Ps2-A2, e o isolado Ps1-A2 apresentou simultaneamente os genes blaKPC e blaVIM. A concomitância desses genes merece destaque pelo acúmulo desses mecanismos genéticos de resistência em uma mesma espécie bacteriana. Conclusão: Os genes para carbapenemases detectados nos isolados de P. aeruginosa, revelam o grande potencial dessa espécie em adquirir e consequentemente disseminar esses mecanismos de resistência no ambiente hospitalar. Precisamos considerar isso como um grande problema de saúde pública, levando em conta as limitações terapêuticas para tratar as infecções causadas por essa espécie. Sendo assim, devemos ressaltar a importância da detecção dessas enzimas nos laboratórios de microbiologia para que sejam adotadas medidas de controle e prevenção da disseminação desses microrganismos multirresistentes(3).

scholarly journals A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

Micromachines ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 472 ◽  
Author(s):  
Yonghee Kim ◽  
Jinyeop Lee ◽  
Sungsu Park
Keyword(s):  
Genomic Dna ◽  
Molecular Detection ◽  
Three Dimensional ◽  
Immunomagnetic Separation ◽  
Automated System ◽  
Dna Purification ◽  
Clinical Samples ◽  
Silica Bead ◽  
3D Printed ◽  
Magnetic Silica

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.

Optimization of DNA isolation and PCR parameters for RAPD analysis of Gonyostomum semen (Raphidophyceae)

2012 ◽  
Vol 18 (1) ◽  
pp. 40-45
Author(s):  
Elena Servienė ◽  
Irena Kemežienė ◽  
Jūratė Kasperovičienė ◽  
Brigita Čapukoitienė ◽  
Vida Rančelienė ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Genetic Variability ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Rapd Analysis ◽  
Dna Purification ◽  
High Quality ◽  
Purification Method ◽  
Rapd Pcr ◽  
Gonyostomum Semen

Abstract Servienė E., Kemežienė I., Kasperovičienė J., čapukoitienė B., Rančelienė V., Koreivienė J., 2012: Optimization of DNA isolation and PCR parameters for RAPD analysis of Gonyostomum semen (Raphidophyceae) [DNR izoliavimas ir PGR parametrų optimizavimas Gonyostomum semen (Raphidophyceae) dumblių RAPD analizei]. - Bot. Lith., 18(1): 40-45. The genomic DNA purification method for Gonyostomum semen algae was optimized by applying different DNA purification techniques and rational modifications. This method allowed to obtain high quality DNA preparations suitable for the phylogenetic analysis and genetic variability investigation of algae. DNA isolated by this method yielded strong and reliable amplification products showing their applicability for RAPD-PCR using random decamer primers. In the present study, the RAPD protocol was optimized for the evaluation of Gonyostomum biodiversity.

scholarly journals Quantitative evaluation of DNA from the tooth pulp exposed to varying temperatures

2016 ◽  
Vol 06 (03) ◽  
pp. 06-09
Author(s):  
Aisha N. Ibrahim ◽  
Vinaya Bhat ◽  
Shilpa M. Shenoy ◽  
Veena A. Shetty
Keyword(s):  
Human Body ◽  
Genomic Dna ◽  
High Speed ◽  
Forensic Identification ◽  
Dna Purification ◽  
Tooth Pulp ◽  
Oral Temperature ◽  
Double Beam ◽  
Significant Difference ◽  
Fire Accidents

Abstract Context: During natural calamities like fire accidents, many times the human body gets charred beyond recognition. Tooth, being the hardest structure in the human body protects the pulp within from such accidents. Forensic identification through the pulpal DNA could be avery useful tool in such situations. Aim: This study was designed to evaluate the quantity of DNA obtained from the pulp when exposed to varying temperatures. Methods and Material: Extracted teeth were subjected to the following temperatures:-80°C,37°C,100°C, 200°C, 300°C, 500°C and 1000°C. Pulp from these teeth were retrieved by horizontally sectioning the teeth using a fine needle diamond point in a high speed air rotor, followed by extraction of DNA with the HipuraA™ Forensic Genomic DNA purification spink it. The samples were quantified using personal computer(PC) based double beam spectro photo meter. Kruskal Wallis test and Mann WhitneyU test were used for statistical analysis. Results: Statistically significant difference (p<0.005) was seen in the quantity of DNA obtained from pulp subjected to higher temperatures as compared to oral temperature. However, there was no significant difference (p>0.05) in the quantity of DNA obtained from teeth subjected to-80°C Conclusions: Increase in temperature decreases the amount of DNA from the tooth pulp where as a decrease in temperature does not cause any change in the quantity of DNA.

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