FindingNemo Extraction 2: Phenol-free Method v2

This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using a phenol-free extraction method. A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters. Kit-free, phenol-free protocol is a modification of NEB's Monarch HMW DNA Extraction Kit for Cells & Blood, with the option to use SDS or CTAB in the lysis buffer. This protocol also uses glass beads for DNA precipitation matrix. We tested this sub-protocol in human cell line, with input cells of 3 millions but can be varied from 1-5 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.

FindingNemo Library 3: KrazyStarFish (KSF) v1

This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit. We named this protocol KrazyStarFish (KSF). It offers a different approach to UL library prep, by using filter paper shaped as a starfish at the DNA precipitation/clean-up step. It can consistently produced N50 > 100 kb with the right transposase to DNA ratio. This protocol was developed by John Tyson at UBC, Vancouver.

Isopropanol DNA Precipitation - Best for DNA Concentration v1

Protocol to precipitate extracted DNA from an aqueous solution to increase concentration or resolubilize in a different storage buffer. *Use isopropanol DNA precipitation if your DNA is suspended in a very large volume, if your DNA concentration is low, or you are trying to concentrate large molecular weight DNA fragments and remove smaller fragments. *Use ethanol DNA precipitation if you are trying to remove salt contamination or precipitate small DNA fragments. Additional Resources: New England Biolabs, "DNA Precipitation: Ethanol vs. Isopropanol". June 23, 2015 Green, Michael R. and Joseph Sambrook, "Precipitation of DNA with Isopropanol". doi:10.1101/pdb.prot093385Cold Spring Harb Protoc2017. Qiagen, "How can I precipitate genomic DNA using isopropanol?".

A reassessment of several erstwhile methods for isolating DNA fragments from agarose gels

Molecular biology research often requires extraction of DNA fragments from agarose gels. In the past decades, there have been many methods developed for this purpose. Currently most researchers, especially novices, use commercial kits for this extraction, although these kits cost money and the procedures involved are not necessarily easier than some erstwhile methods. We herein reintroduce and reassess several simple and cost-free older methods. One method involves excising a slice of the gel containing the DNA fragment, followed by a thaw-and-freeze procedure to release the DNA from the gel slice into the gel-making buffer. The second method involves a dialysis tubing and requires electroelution of the DNA from the gel slice in the tubing. The third one is to centrifuge the gel slice to release the DNA. The fourth method requires electro-transfer of the DNA from the gel into a filter paper, while the fifth one includes either allowing the DNA in the slice to be dissolved into a buffer or dissolving the DNA-containing gel slice, followed by DNA precipitation with ethanol or isopropanol. The strengths and weaknesses of these methods are discussed to assist researchers in making their choice. We also point out that some of the end uses of the DNA fragment in the agarose gel may not actually require extraction of the DNA. For instance, a tiny DNA-containing gel block or filter paper can be directly used as the template in a nested or semi-nested polymerase chain reaction to preliminarily determine the identity of the DNA fragment.

An optimized DNA extraction protocol for wood DNA barcoding of Pterocarpus erinaceus

The isolation of wood DNA is a crucial step in the process of genetic identification of wood tissues and the current wood DNA extraction method is a limiting factor. For some valuable wood samples sent for forensic identification, the size of allowable sample is limited. Additionally, the identification process is so lengthy that it often cannot meet the needs of law enforcement. This study describes an optimized protocol that minimizes the sample size and duration of DNA extraction without decreasing the yield of DNA. Experiments on sample mass per extraction, sample lysis time and DNA precipitation time were carried out by a series of gradient tests. The amounts of DNA extracted were evaluated by the copy numbers of target DNA barcodes (rbcL and matK) from droplet digital PCR (ddPCR). It is the first to apply ddPCR technology to quantify and evaluate accurately DNA extracted from wood. The results indicated that 300 mg is an optimal sample mass when keeping the volume of DNA lysis buffer constant, which reduces the sample usage by 40%. Five hours is the optimal sample lysis time. Extending the duration of DNA precipitation does not significantly increase the amplicon yield from wood specimens of Pterocarpus erinaceus. The protocol developed in this study shortens the period of DNA extraction from wood tissues by approximately 58%. The amplicon yields obtained using the optimized method in this study indicate good extraction efficiency, and the wood samples sent for certification were identified as Pterocarpus erinaceus using the barcode combination matK+ndhF-rpl32+ITS2. This method will be suitable for the broad applicability of DNA identification and conservation of global wood resources.

The Nuclear Receptor COUP-TFII Regulates Amhr2 Gene Transcription via a GC-Rich Promoter Element in Mouse Leydig Cells

The nuclear receptor chicken ovalbumin upstream promoter–transcription factor type II (COUP-TFII)/NR2F2 is expressed in adult Leydig cells, and conditional deletion of the Coup-tfii/Nr2f2 gene impedes their differentiation. Steroid production is also reduced in COUP-TFII–depleted Leydig cells, supporting an additional role in steroidogenesis for this transcription factor. COUP-TFII action in Leydig cells remains to be fully characterized. In the present work, we report that COUP-TFII is an essential regulator of the gene encoding the anti-Müllerian hormone receptor type 2 (Amhr2), which participates in Leydig cell differentiation and steroidogenesis. We found that Amhr2 mRNA levels are reduced in COUP-TFII–depleted MA-10 Leydig cells. Consistent with this, COUP-TFII directly activates a −1486 bp fragment of the mouse Amhr2 promoter in transient transfection assays. The COUP-TFII responsive region was localized between −67 and −34 bp. Chromatin immunoprecipitation assay confirmed COUP-TFII recruitment to the proximal Amhr2 promoter whereas DNA precipitation assay revealed that COUP-TFII associates with the −67/−34 bp region in vitro. Even though the −67/−34 bp region contains an imperfect nuclear receptor element, COUP-TFII–mediated activation of the Amhr2 promoter requires a GC-rich sequence at −39 bp known to bind the specificity protein (SP)1 transcription factor. COUP-TFII transcriptionally cooperates with SP1 on the Amhr2 promoter. Mutations that altered the GCGGGGCGG sequence at −39 bp abolished COUP-TFII–mediated activation, COUP-TFII/SP1 cooperation, and reduced COUP-TFII binding to the proximal Amhr2 promoter. Our data provide a better understanding of the mechanism of COUP-TFII action in Leydig cells through the identification and regulation of the Amhr2 promoter as a novel target.

A RADAR-Based Assay to Isolate Covalent DNA Complexes in Bacteria

Quinolone antibacterials target the type II topoisomerases gyrase and topoisomerase IV and kill bacterial cells by converting these essential enzymes into cellular poisons. Although much is known regarding the interactions between these drugs and enzymes in purified systems, much less is known regarding their interactions in the cellular context due to the lack of a widely accessible assay that does not require expensive, specialized equipment. Thus, we developed an assay, based on the “rapid approach to DNA adduct recovery,” or RADAR, assay that is used with cultured human cells, to measure cleavage complex levels induced by treating bacterial cultures with the quinolone ciprofloxacin. Many chemical and mechanical lysis conditions and DNA precipitation conditions were tested, and the method involving sonication in denaturing conditions followed by precipitation of DNA via addition of a half volume of ethanol provided the most consistent results. This assay can be used to complement results obtained with purified enzymes to expand our understanding of quinolone mechanism of action and to test the activity of newly developed topoisomerase-targeted compounds. In addition, the bacterial RADAR assay can be used in other contexts, as any proteins covalently complexed to DNA should be trapped on and isolated with the DNA, allowing them to then be quantified.