High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application

The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C. elegans has, however, been challenging because of the worms’ thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.     

High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer

Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.     

High Throughput BAC DNA Isolation for Physical Map Construction of Sorghum (Sorghum bicolor)

With the aim of constructing a physical map of sorghum, we developed a rapid, high throughput approach for isolating BAC DNA suitable for restriction endonuclease digestion fingerprinting, PCR- based STS-content mapping, and BAC-end sequencing. The system utilizes a programmable 96 channel liquid handling system and associated accessories that permit bacterial cultivation and DNA isolation in 96-well plate format. This protocol details culture conditions that optimize bacterial growth in deep-well plates and criteria for BAC DNA isolation to obtain high yields of quality BAC DNA. The system is robust, accurate, and relatively cost-effective. The BAC DNA isolation system has been tested during efforts to construct a physical map of sorghum.     

High throughput isolation of DNA and RNA in 96-well format using a paint shaker

Most steps in plant nucleic acid isolation are easily adapted to 96-well format; however, tissue disruption typically is performed on samples individually and often is the rate-limiting step in sample processing. We have found that DNA and RNA isolation from Arabidopsis tissue can be carried out in 96-well format using a paint shaker fitted with an adapter for tissue disruption.     

An efficient, automatable template preparation for high throughput sequencing

We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.     

Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes

To meet the new challenge of generating the draft sequences of mammalian genomes, we describe the development of a novel high throughput 96-well method for the purification of plasmid DNA template using size-fractionated, acid-washed glass beads. Unlike most previously described approaches, the current method has been designed and optimized to facilitate the direct binding of alcohol-precipitated plasmid DNA to glass beads from alkaline lysed bacterial cells containing the insoluble cellular aggregate material. Eliminating the tedious step of separating the cleared lysate significantly simplifies the method and improves throughput and reliability. During a 4 month period of 96-capillary DNA sequencing of the Rattus norvegicus genome at the Baylor College of Medicine Human Genome Sequencing Center, the average success rate and read length derived from >1 800 000 plasmid DNA templates prepared by the direct lysis/glass bead method were 82.2% and 516 bases, respectively. The cost of this direct lysis/glass bead method in September 2001 was ~10 cents per clone, which is a significant cost saving in high throughput genomic sequencing efforts.     

An efficient,simple and high throughput protocol for cotton genomic DNA isolation

Cotton is a stubborn plant for genomic DNA isolation due to its high-level of polyphenolics, polysaccharides and secondary metabolites. Genomics and molecular biology studies require high quality and large quantity of DNA. We have standardized an efficient miniprep protocol for cotton genomic DNA isolation, which not only provides higher yield from 800 to 1400 µg of DNA from 200 to 300 mg of fresh leaf tissue but also provide excellent purity. The DNA is amenable to all elementary enzymatic preparations, PCR techniques, Southern blotting and to high end genomic studies. The technique does not require liquid nitrogen, needs small amount of sample, less time, fewer chemicals and one can process up to 100 samples per day. The genomic DNA extracted was good for transgenic event characterization and marker assisted selection.     

Evaluation of a reliable and cost-effective method of DNA isolation for mouse genotyping

Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, “crude” DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.     

Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ～100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.     

A strategy for recovering high quality genomic DNA from a large number of Phytophthora isolates

We present a strategy to recover high molecular weight genomic DNA from large numbers of isolates of Phytophthora. Included are steps for generating mycelial mass in 24-well reuseable deep well plates, efficient lyophilization and disruption of the mycelium and genomic DNA extraction with 96-well glass fiber filter plates. The resulting DNA is consistently high molecular weight and is suitable for applications that require high quality DNA such as AFLP analysis and TILLING. A single operator easily can manage mycelium preparation and/or DNA extraction from 384 isolates in a single day and this approach might be useful for other fungi or fungi-like organisms that can be grown in liquid media.     

Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts

Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.     

High-throughput DNA extraction method suitable for PCR

PCR has become one of the most popular techniques in functional genomics. Projects in both forward and reverse genetics routinely require PCR amplification of thousands of samples. Processing samples to extract DNA of sufficient purity for PCR is often a limiting step. We have developed a simple 96-well plate-based high-throughput DNA extraction method that is applicable to many plant species. The method involves a simple incubation of plant tissue samples in a DNA extraction buffer followed by a neutralization step. With the addition of a modified PCR buffer, the extracted DNA enabled the robust amplification of genomic fragments from samples of Arabidopsis, tobacco, sorghum, cotton, moss, and even pine needles. Several thousand DNA samples can be economically processed in a single day by one person without the use of robotics. This procedure will facilitate many technologies including high-throughput genotyping, map-based cloning, and identification of T-DNA or transposon-tagged mutants for known gene sequences.     

Multichannel plating unit for high-throughput plating of cell cultures

High-throughput genomic approaches to gene function or target identification have led to the development and implementation of the 96-well format for many standard molecular biology manipulations. The apparatus described here, a Multichannel Plating Unit, is designed to plate out individual cultures efficientlyfrom standard 96-well culture blocks. Following transformation, aliquots of culture are loaded onto sterile beads that are rolled along individual channels of agar media. After the beads traverse the channel, they drop into the exit alley for disposal via an exit pore. The apparatus presented has 12 individual lanes, and the spacing is compatible with a standard 12-channel pipettor Thus, the unit allows for the rapid plating of 12 individual cultures at a time. For one 96-well block of transformants, this method reduces the labeling and plating effort from 96 culture dishes that are spread individually to eight multichannel plates. The savings in time, materials, and storage space is significant     

An automated apparatus for the real-time monitoring of bioluminescence in plants

We developed an automated, high-throughput, bioluminescence-monitoring apparatus that can monitor 1920 individual plant seedlings under uniform light conditions. The apparatus is composed of five units: (i) a plate platform that can hold 20 96-well microplates under uniform light conditions, (ii) a scintillation counter, (iii) a robot that conveys plates between the plate platform and a scintillation counter, (iv) a sequence controller, and (v) an external computer that collects and analyzes bioluminescence data automatically. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms; neither was affected by the well position in a plate or the plate position on the platform. The apparatus is a powerful tool for both large-scale detailed analysis of gene expression and large-scale screening of mutants.     

小麦—簇毛表6VS／6AL易位系可转化人工染色体（TAC）文库的构建

可转化人工染色体（Ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｃｏｍｐｅｔｅｎｔ Ａｒｔｉｆｉｃｉａｌ Ｃｈｒｏｍｏｓｏｍｅ,ＴＡＣ）是具有克隆和转移大片段基因能力的新型载体,是植被基因克隆和转化的有效工具。为了克隆泪科抗白粉病基因和其它基因,本研究用ＴＣＡ载体ｐＹＬＴＡＣ１７构建了带有抗白粉病基因Ｐｍ２１的小麦＝簇毛麦６ＶＳ／６ＡＬ易位系的基因组ＤＮＡ文库。该文库包含２１０万个克隆平均插入征段３５ｌｂ,相当于     

High-throughput SNP genotyping on universal bead arrays

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Rapid method for DNA isolation from a tough cell wall green alga <Emphasis Type="Italic">Tetraspora</Emphasis> sp. CU2551

Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30–105 s at 70 °C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains.     

Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probes

A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10–1.98 and 4.43– 12.54 fmole ml⁻¹ homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.