We would like to thank the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund, Genome British Columbia and the British Columbia Ministry of Forests and Range for supporting ongoing studies of forest soil productivity. S.L was supported by a fellowship from the TULA foundation funded Centre for Microbial Diversity and Evolution.

Part I: Extraction of DNA
Procedure
1. 8 hours are required, for processing 6 samples excluding quantification of DNA.
Before you start the extraction, you will need to pre-chill mortar, pestle and spatula in a -20 &deg;C freezer or using liquid nitrogen. Also, pre-chill 50 ml tubes containing 20 ml chloroform isoamyl alcohol (24:1) on ice. Set the hybridization oven to 65 &deg;C to pre-heat. Check CTAB solution, if it is crystallized warm to 60 &deg;C to melt the crystals. Complete the denaturing solution and extraction buffer by adding the last ingredients just before you start the extraction (see the recipe below). Conduct all extraction procedures in a fume hood.
2. Prepare the denaturing buffer and extraction buffer. Note: To maximize the DNA yield, it is critical to precisely make buffers following the instructions. 
3. Place 2 g of soil in a pre-chilled mortar. Note: Soil collected in the field should be frozen on dry ice for transport and stored at -80 &deg;C. Thaw and sieve soil using a 2 mm sieve prior to DNA extraction. You can increase the starting amount of soil up to 10 g.
4. Add 1 ml of denaturing solution to the soil.
5. Freeze and grind the soil in liquid N2 using a pestle until the soil particles look powdery and homogenous. Repeat freezing and grinding twice. Note: Keep the soil sample frozen during the grinding, using enough liquid nitrogen to fully cover the soil sample. Slight melting during the grinding is acceptable.
6. Transfer the ground soil to a 50 ml conical tube using a pre-chilled spatula. Note: The ground soil sample can be stored at -80 &deg;C at this point, if necessary. 
7. Add 9 ml of extraction buffer. To mix the solution and soil, vortex briefly at low speed. Note: To ensure the buffer is homogeneous, warm it at 60 &deg;C for 10-15 minutes before use. Keep the rest of buffer at 60 &deg;C throughout the extraction. Vortex at speed 8 (on a scale where 10 is the maximum). 
8. Incubate for 40 min at 65&deg;C in a hybridization oven. During the incubation, continuously rotate the tubes at the lowest speed, or gently invert the tubes every 10 min. Note: Place the tube horizontally in a hybridization oven to allow buffer to contact soil over a large surface area. The solution may look a bit viscous during incubation.
9. Centrifuge at 1800 xg, for 10 min at 10 - 14 &deg;C. Transfer supernatant into the pre-chilled 50 ml conical tube containing 20 ml chloroform-isoamyl alcohol (24:1), keep on ice. Note: When you transfer supernatant, be careful not to disturb the organic layer (bottom layer). Leave 1-2 ml of supernatant if it is necessary in order to collect only clean supernatant. You often can see a beige layer (approximately 1-3 mm in thickness) on top of the supernatant. Do not disturb this layer when collecting the supernatant. Try to slide the tip along the tube wall to prevent breakage of this beige layer. If the supernatant is too turbid, repeat the chloroform-isoamyl alcohol extraction one more time on the supernatant. Do not use disposable plastic pipettes when transferring chloroform, as chloroform will melt the plastic. Use a glass pipette, or a glass graduated cylinder, or pour chloroform directly into 50 ml conical tubes.
10. Extract the soil pellets 2 more times;
10.1. Add 5 ml extraction buffer to the soil pellets, gently mix using 1 ml tip followed by brief vortexing at low speed. Note: Shake and mix the buffer before use. 
10.2. Incubate at 65&deg;C for 10 min in hybridization oven. Centrifuge at 1800 xg, for 10 min at 10 - 14 &deg;C. Transfer supernatant to the tube at step 9. Keep the tube with collected supernatant and chloroform-isoamyl alcohol on ice during the extraction. Note: Make sure you add the supernatant to the same tube without cross mixing between the samples.
10.3. Repeat 9.1 and 9.2 one more time.
11. Using a rotating plate, very gently rock the tube that contains the collected supernatant (14-19 ml) and chloroform-isoamyl alcohol at 1.25 rpm for 10 min. Note: Close the lid very tightly to prevent leakage. Lay a paper towel on the rocking plate before you place the tube so any leaks can be readily detected. 
12. Centrifuge at 1800 xg, for 20 min at 10 - 14 &deg;C.
13. Transfer aqueous phase to a new tube. Note: Do not disturb the interphase between the aqueous phase (top layer) and organic layer (bottom layer) when you transfer the supernatant. Leave approximately 1.5- 2 ml supernatant to ensure collection of only clean supernatant.
14. Recover the DNA using isopropyl alcohol (step 15.a - 21.a). Alternatively, use Amicon&reg; Ultra-15 Centrifugal Filter Devices (step 15.b - 21.b) if you expect very low amount of biomass in your sample, which will produce hardly visible pellet by isopropyl alcohol precipitation that is difficult to recover.
15.a. Place the supernatant to a new tube ultracentrifuge.
16.a. Add isopropyl alcohol to the collected supernatant at a volume ratio of 0.6:1. Invert the tubes gently a few times to mix.
17.a Incubate for 30 min at room temperature.
18.a. Centrifuge at 16000 xg, for 20 min at 20 - 25 &deg;C. Ensure the weights of all the tubes are balanced well before the centrifugation step. Note: Mark the direction of centrifugal force on the tube, so you can detect the DNA pellet more easily when the DNA yield is low.
19.a. Remove isopropyl alcohol by decanting or vacuum suctioning. Be careful not to disturb the DNA pellet. Note: Remove isopropyl alcohol as soon as centrifugation is completed. Be very careful not to lose the DNA pellet. The size of DNA pellet can vary widely depending on the soil type, from a clearly visible (~9 mm diameter) to hardly visible. It is recommended to remove the residue of isopropyl alcohol by suctioning along the wall of the tube carefully so as not to suction out the DNA pellet. 
20.a. Dry DNA pellet at room temperature for 5 - 20 min. Note: Do not over dry DNA pellet, otherwise it will be difficult to redissolve the DNA.
21.a. Resuspend the DNA pellet in 200 &mu;l TE. Mix it by gentle tapping. Transfer the DNA solution into 1.5 ml tube. Keep the tube at 4&deg;C overnight in order to fully dissolve DNA. Note: Depending on the size of pellet, you can adjust the volume of TE to add. Usually 200 - 400 &mu;l of TE works well.
*Alternatively follow the steps 15.b - 21.b.
15.b. Pre-wash an Amicon&reg; Ultra-15 Centrifugal Filter Devices; add 15 ml TE to the device and centrifuge at 3500 xg, for 10 min. Discard the flow-through.
16.b. Place the DNA solution from step 13 to the pre-washed Amicon filter. Centrifuge at 3500 xg until the DNA volume is reduced to 200 - 500 &mu;l. Discard the flow-through.
17.b. Wash the DNA with TE twice; add 10 ml TE to the Amicon filter devices. Centrifuge at 3500 xg until the DNA volume is reduced to approximately 200 &mu;l. Discard the flow-through. Repeat one more time.
18.b. Transfer the DNA solution on the filter to a new 1.5 ml tube. 
19.b. Add 40 &mu;l TE to the Amicon filter. Wash both side of filter membranes by pipetting up and down in order to recover any DNA residue on the filter. Add this wash solution to the tube at step 18.b.
20.b. If necessary, the DNA can be further concentrated by Microcon&reg; Ultracel YM-30 Centrifugal Filter Unit; pre-wash the filter by adding 200 &mu;l TE and centrifuging at 10000 xg for 7 minutes. Add the DNA solution to the filter and centrifuge at 5000 xg for 1 min. Repeat centrifugation until the amount of DNA solution on the filter reduced to 50 &mu;l approximately. Note: Maximum initial sample volume for Microcon filter is 500 &mu;l. Do not centrifuge at speeds over 14000 x g. Do not over centrifuge until the membrane dries completely. Ensure that some liquid still covers the filter after centrifugation. If you over centrifuged and the membrane is dry, then add 15 &mu;l TE, agitate gently for 30 seconds, and proceed to step 21.b.
21.b. Place the filter unit upside down in a new Microcon tube and centrifuge at 1000 x g for 3 min to recover the DNA. 
22. Quantify DNA by TAE gel (0.8%, 0.5xTAE, 50 V for 4 hours), and by Nanodrop spectrophotometer or Pico-green assay and plate reader. Note: DNA amount can be estimated by comparing the sample band to the marker band with known concentration (High molecular DNA marker or &#955;HindIII) on the gel. 
23. Check the integrity of high molecular weight DNA using Pulsed Field Gel Electrophoresis (PFGE) with 1%, 1xTAE gel (For more details see the Step II, part2 at <a href="https://www.jove.com/index/details.stp?id=1387">https://www.jove.com/index/details.stp?id=1387</a>, doi: 10.3791/1387 by Taupp et al. 2009). Note: You can use regular agarose gel for this QC purpose instead of low melting agarose. In brief, electrophoresis conditions are as follows: 2-250 kb, 6 volts, initial switching time: 5 sec, final switching time: 15 sec, run for 12 hrs at 14 &deg;C. SYBR Gold staining for one hour to visualize the DNA on the gel. 
24. If the molecular weights of extracted DNA are satisfactory, then proceed to the following CsCl gradient centrifuge step. Alternatively, DNA can be stored at -20 &deg;C or -80 &deg;C before the CsCl centrifuge. The median size of the DNA should be the equal or exceed 36 Kb, if you want to construct a large insert metagenomic DNA library. 
Part II. DNA purification using cesium chloride gradient ultracentrifugation
Preparation of centrifuge: 1.5 - 2 hours Centrifugation step: 18 hours Removing ethidium bromide (EtBr) and concentrating DNA after centrifugation: 3 hours
Procedure
For details, refer Wright et al. 2009. <a href="https://www.jove.com/index/details.stp?id=1352">https://www.jove.com/index/details.stp?id=1352</a>, doi: 10.3791/1352
Representative Results/Outcome
The total amount of genomic DNA isolated from 2 g of forest soil organic (O horizon) and mineral horizons (Ae, AB, and Bt horizons) (sampled from the Long Term Soil Productivity (LTSP) site located at Skulow lake, BC, Canada and managed by BC Ministry of Forests and Range) ranged from 10 mg to 180 mg. The median size of isolated DNA was usually between 40 - 60 kb (Figure 1). The ratio of absorbance at 260 nm/280 nm was between 1.3-1.6 and there was a peak at 230 nm, which may indicate humic acid contamination often observed in soil samples. The CsCl gradient centrifugation drastically reduced this contamination and also increased the ratio of 260/280 to 1.8 - 1.9 as shown in Figure 2a and 2b.
<img src="/files/ftp_upload/1569/1569fig1.jpg" alt="Figure 1" /> Figure 1. PFGE pictures of the extracted genomic DNA before CsCl ultracentrifugation. lane1: 1 kb marker 100 ng, lane 2: &lambda;Hind III marker 100 ng, lane 3: &lambda;Hind III marker 250 ng, lane 4: &lambda;Hind III marker 500 ng, lane5: Fosmid 36 kb marker 100 ng, lane 6 and 7: genomic DNA isolated from the mineral soil horizon Ae, lane 8 and 9: genomic DNA isolated from the mineral soil horizon AB at the LTSP site located Skulow lake, BC 
<img src="/files/ftp_upload/1569/1569fig2tmb.jpg" alt="Figure 2" border="0" /> Figure 2. Chromograms of genomic DNA detected by spectrophotometer before (A) and after CsCl ultracentrifugation (B). Note the reduction in the absorbance value at the 230 nm wavelength after CsCl ultracentrifugation, which indicates improvement in the genomic DNA quality. Please <a href="https://www.jove.com/files/ftp_upload/1569/1569fig2.jpg">click here</a> to see a larger version of figure 2.

<ol>
	<li>Hurt, R. A., Qiu, X., Wu, L., Roh, Y., Palumbo, A. V., Tiedje, J. M., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recovery+of+RNA+and+DNA+from+soils+and+sediments.">Simultaneous recovery of RNA and DNA from soils and sediments.</a> AEM. 67 (10), 4495-4503 (2001).</li><li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+extraction+from+0.22+&#956;M+Sterivex+filters+and+cesium+chloride+density+gradient+centrifugation.">DNA extraction from 0.22 &#956;M Sterivex filters and cesium chloride density gradient centrifugation.</a> JoVE. , (2009).</li><li>Taupp, M., Lee, S., Hawley, A., Yang, J., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+insert+environmental+genomic+library+production.">Large insert environmental genomic library production.</a> J Vis Exp. , (2009).</li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
			<div>
			Recipe 
* Denaturing solution 
 4 M Guanidine isothiocyanate 
 10 mM Tris-HCl (pH 7.0) 
 1 mM EDTA 
 0.5% 2-mercaptoethanol
<table border="0" style="font-size:9pt; width:400px;">
<tr><td colspan="2">Denaturing solution: 10 ml in total </td></tr>
<tr><td>Guanidine isothiocyanate (MW 118.16)</td><td>4.73 g</td></tr>
<tr><td>1M Tris-HCl (pH 7.0)</td><td>100 &mu;l</td></tr>
<tr><td>0.5M EDTA</td><td>20 &mu;l</td></tr>
<tr><td colspan="2">Add water to 9.95 ml in total.</td></tr>
<tr><td colspan="2">Autoclave.</td></tr>
<tr><td colspan="2">Add 50 &mu;l 2-mercaptoethanol just before use. (5 &mu;l of 2-mercaptoethanol per 1 ml Denaturing Solution) 
Note: Keep the Denaturing solution at 4 &deg;C.&nbsp; Do not use buffer older than one week. If possible, make fresh buffer to use.</td></tr>
</table>
**Extraction Buffer 
 100 mM Sodium phosphate buffer [pH 7.0] 
 100 mM Tris-HCl [pH 7.0] 
 100 mM EDTA [pH 8.0] 
 1.5 M&nbsp; NaCl 
 1% Hexadecyltrimethylammonium bromide (CTAB) 
 2% SDS
<table border="0" style="font-size:9pt; width:400px;">
<tr><td colspan="2">Extraction Buffer for 1 L in total</td></tr>
<tr><td>1M Sodium phosphate buffer [pH 7.0]*</td><td>100 ml</td></tr>
<tr><td>1M Tris-HCl [pH 7.0]</td><td>100 ml</td></tr>
<tr><td>0.5M EDTA [pH 8.0]</td><td>200 ml</td></tr>
<tr><td>5 M NaCl</td><td>300 ml</td></tr>
<tr><td colspan="2">Autoclave and keep it at room temperature.</td></tr>
<tr><td colspan="2">Add 200ml, 5% Hexadecyltrimethylammonium bromide (CTAB, autoclaved) and 100 ml, 20% SDS (autoclaved) just before use. If CTAB was crystallized, melt it at 60 &deg;C. 
 * 1M Sodium phosphate buffer: 57.7 ml, 1M Sodium phosphate monobasic (NaH2PO4) and 42.3 ml, 1M Sodium phosphate dibasic (NaH2PO4). Adjust pH to 7.0 
Note: Do not use 20 % SDS if it has precipitation. It is normal to see milky suspension when you add SDS to the solution. Once you add SDS, place the extraction buffer at 60 &deg;C to ensure SDS is well suspended.</td></tr>
</table>		</div>

extraction of high molecular weight genomic dna from soils and sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

Watch this Scientific Journal Video about Extraction of High Molecular Weight Genomic DNA from Soils and Sediments at JoVE.com

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

extraction-high-molecular-weight-genomic-dna-from-soils

Nanyang Technological University

Research

JoVE Journal

Biology

25.7K Views.  University of British Columbia - UBC. A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described. 

Video: Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Isolation of Genomic DNA from Mouse Tails

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...