MODIFIKASI METODE ISOLASI DNA CETYL TRIMETHYLAMMONIUM BROMIDE (CTAB) UNTUK SAMPEL EPITEL PIPI MANUSIA

Isolation of deoxyribonucleotide (DNA) is an important step in molecular analysis. In this process, DNA must be obtained in sufficient quantities and in good quality for any further analysis. The Cetyl Trimethylammonium Bromide (CTAB) method is commonly used in DNA isolation of plant or fungal. This method is an alternative in DNA isolation since it is easy and inexpensive. This study aims to modify the CTAB method for DNA isolation from human cheek epithelium for any molecular analysis. Epithelial cells were taken from the oral cavity of the researcher. The isolation protocol included cell lysis step with CTAB buffer and proteinase-K, purification step with the addition of chloroform:isoamylalcohol (24:1), precipitation step with isopropanol. The results of the ratio analysis of DNA spectrophotometer at wavelengths of 260 and 280 nm in the range of 1.73-1.85. The quality of DNA isolation was observed by agarose gel electrophoresis and a firm band was obtained after Ethidium Bromide staining. The DNA concentration in both methods ranged from 400-480 mg/mL. The time required for both methods ranges from 2.5-3 hours. The modified CTAB method DNA isolation protocol produces DNA that has good quality and quantity for molecular analysis processes, such as Polymerase Chain Reaction (PCR).

Chromosome-level de novo assembly of Coprinopsis cinerea A43mut B43mut pab1-1 #326 and genetic variant identification of mutants using Nanopore MinION sequencing

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.HighlightA chromosome-level genome assembly of C. cinerea #326A fast and efficient high-molecular-weight fungal genomic DNA isolation protocolStructural variant and single nucleotide variant calling using Nanopore readsA series of solutions and reference parameters for fungal genomic analysis on MinION

The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

ABSTRACTThe commercialisation of miniprep kits supplanted the original alkaline lysis method for plasmid DNA preparation, and had remained relatively unchanged for almost two decades. The Miraprep substantially improved the yields of miniprep kits. However, the method still relies on commercial kits, which can be a burden financially to certain projects. Additionally, Pronobis et al. also identified loss of RNAse activities in miniprep kits over time. The present novel plasmid DNA isolation protocol addresses the two issues mentioned above utilising alkaline lysis and alkaline hydrolysis principles. With a largely identical workflow and operation time, the Macerprep will significantly reduce costs of establishing new laboratories as well as maintenance of running molecular biology laboratories.

A Robust DNA Isolation Protocol from Filtered Commercial Olive Oil for PCR-Based Fingerprinting

Extra virgin olive oil (EVOO) has elevated commercial value due to its health appeal, desirable characteristics and quantitatively limited production, and thus it has become an object of intentional adulteration. As EVOOs on the market might consist of a blend of olive varieties or sometimes even of a mixture of oils from different botanical species, an array of DNA-fingerprinting methods have been developed to check the varietal composition of the blend. Starting from a comparison between publicly available DNA extraction protocols, we set up a timely, low-cost, reproducible and effective DNA isolation protocol, which allows an adequate amount of DNA to be recovered even from commercial filtered EVOOs. Then, in order to verify the effectiveness of the DNA extraction protocol herein proposed, we applied PCR-based fingerprinting methods starting from the DNA extracted from three EVOO samples of unknown composition. In particular, genomic regions harboring nine simple sequence repeats (SSRs) and eight genotyping-by-sequencing-derived single nucleotide polymorphism (SNP) markers were amplified for authentication and traceability of the three EVOO samples. The whole investigation strategy herein described might favor producers in terms of higher revenues and consumers in terms of price transparency and food safety.

Effective Molecular Identification of Ectomycorrhizal Fungi: Revisiting DNA Isolation Methods

A better understanding of ectomycorrhizal symbiosis leads to numerous advancements in forest management and environmental protection. The morphological identification of the ectomycorrhizae often proves to be misleading. For this reason, in order to study the ectomycorrhizal fungi communities, a number of molecular methods that require the isolation of nucleic acids are being used. However, ectomycorrhizal root tips, low mass heterogenic material rich in inhibitors, are a recalcitrant substrate in DNA isolation. It is common for published studies to include some number of unidentified root tips in their results, in spite of diverse isolation protocols being available to researchers. This study aims to analyze the relationship between the collected fungal material and later isolation results, and to propose a DNA isolation protocol specifically optimized for ectomycorrhizal root tips. It was found that the taxonomic position can be used to predict the potential isolation efficiency, with Ascomycota being generally more difficult from which to isolate DNA. After a number of cell lysis and lysate purification methods were evaluated, the joined approach of mechanical and chemical lysis, followed by silica column purification, was found to provide the best results, even with recalcitrant material.

Comparison of detergent and CTAB method for isolation of DNA from Salak ( Salacca zalacca (Gaert.) Voss. ‘Pondoh’)

This study conducted in Laboratory of Molecular Biology, Balai Besar Bioteknologi dan Sumberdaya Genetik Pertanian (BB- Biogen) Bogor. The aims of this study are to determine and comparing the quantity, quality and the efficiency of DNA isolation result using detergent method and CTAB method. The parameters observed in this study are the value of DNA concentration, purity, and visualization result using gel electrophoresis. The samples are the leaves of Salak ‘Pondoh’ (Salacca zalacca (Gaert.) Voss.). Detergent method is a method which was developed by Faculty of Biology UGM, it has simple method and relatively affordable cost. Meanwhile, CTAB method is one of the commonly used methods of DNA isolation protocol with relatively expensive cost. Detergent method used detergent in the cell wall separation and protein removal in the sample. The CTAB method used Cetyltrimethyl ammonium bromide (CTAB) for cell membrane separation in the sample. The research methods included DNA isolation with detergent and CTAB methods, PCR analysis and electrophoresis. Data analysis was done quantitatively using spectrophotometric method and qualitative used electrophoresis method. The result of the study showed that DNA isolation using CTAB method showed higher purity compared with detergent method with the purity values ranging from 1,3- 1,4 . Meanwhile, the concentration of DNA in the detergent method was higher than that of CTAB with the highest concentration of 1730 µg/ml. There is no difference between the quality of genomic DNA isolated by CTAB and detergent methods.

AN OPTIMIZED DNA ISOLATION PROTOCOL ENABLES AN INSIGHT INTO MOLECULAR GENETIC BACKGROUND OF ENDEMIC Moltkia petraea (Tratt.) Griseb. FROM BOSNIA AND HERZEGOVINA

The Dinaric endemic plant species Moltkia petraea (Tratt.) Griseb. is often called a "living fossil" of ancient Tertiary flora, with great importance for Bosnia and Herzegovina’s biodiversity. Considering its narrow and limited distribution range, insufficient data on the molecular background of this species is given so far. Due to the presence of various secondary metabolites that interfere with the DNA, isolation of nucleic acids from plant cells is known to be challenging. Even in closely related species it is necessary to optimize DNA isolation protocol in order to obtain high quality PCR amplifiable DNA. We collected 91 samples from five populations in Herzegovina. Doyle and Doyle (1987) CTAB protocol was modified by adding vitamin C (ascorbic acid) to the cell lysis buffer to improve DNA yield and quality. trnL(UAA) intron and nrDNA (ITS1, ITS2) molecular markers were applied to demonstrate amplifiability of isolated DNA and elucidate the intra- and interpopulation genetic diversity. Our results suggest a successful PCR amplification for 81% of the analyzed samples. PCR-RFLP analysis of trnL(UAA) revealed that all individuals in five populations have the same haplotype based on the obtained enzymatic profile for three enzymes (TaqI, HinfI, HindII). Alignment and comparison of ITS sequences didn’t reveal any hypervariable portion that could be informative in elucidating the genetic diversity of M. petraea populations. Further studies with additional application of microsatellite loci, RAPD and AFLP methods are necessary in an attempt to get insights into the genetic diversity of M. petraea.