We acknowledge funding support from the Rockefeller Foundation (Rockefeller Foundation Grant Number 2021 HTH 018) as part of the APSI India team (Alliance for Pathogen Surveillance Innovations&#160;https://data.ccmb.res.in/apsi/team/). We also acknowledge the financial aid provided by Axis Bank in supporting this research and the Trivedi School of Biosciences at Ashoka University for equipment and other support.

1. Wastewater sampling
<ol>
	<li>Dip a 500 mL polypropylene beaker in the open drain or sewage treatment plant (STP) reservoir and collect ~300 mL of wastewater sample.</li>
	<li>Transfer ~250 mL of the sample to a 250 mL autoclaved polypropylene bottle.</li>
	<li>Screw on the cap of the bottle and seal it with a plastic film. Keep the bottle upright in a closed bag.</li>
	<li>Transport the sample upright in a closed container at ambient temperature.</li>
	<li>Heat-inactivate the sample at 70 &#176;C in a hot air oven for 4 h before processing it for DNA extraction.</li>
</ol>
2. DNA extraction from wastewater samples
<ol>
	<li>Once the wastewater sample is cooled, vortex the samples at maximum speed and let the debris/sludge settle.</li>
	<li>Decant 27.5 mL of the wastewater in 50 mL centrifuge tubes and add 13.5 g of PEG-8000 (final concentration 30%) and 3 g of NaCl (final concentration 1.2 M) to it and mix well till completely dissolved.</li>
	<li>Incubate the sample overnight (~16-18 h) at 4 &#176;C.</li>
	<li>Centrifuge the sample at 15,500 x g at 4 &#176;C for 30 min.</li>
	<li>Discard the supernatant. 
	NOTE: PEG-8000 at 30% is highly viscous, and the pellet can get dislodged while discarding the supernatant. The supernatant should be decanted slowly and carefully to ensure the pellet is not lost. Steps 2.6 to 2.23 are performed using the Soil DNA extraction kit according to the kit instructions with a few modifications.</li>
	<li>Dissolve the pellet in 800 &#181;L of the lysis solution of the kit and add the solution to the mixed zirconium bead tube. 
	NOTE: If processing larger volumes, pool the pellets from the desired number of tubes. For example, if processing 160 mL of sample; there will be four 50 mL tubes containing wastewater with PEG and NaCl. After centrifugation of all four tubes at 15,500 x g at 4 &#176;C for 30 min, discard supernatant from all four tubes. Add 200 &#181;L of CD1 solution to each, re-suspend the pellets, and pool them together in one bead tube.</li>
	<li>Secure the bead tube horizontally on a vortex. Vortex the tube at maximum speed for 20-30 min. 
	NOTE: Prolonged vortexing ensures complete lysis and homogenization of the samples, which improves DNA yield.</li>
	<li>Spin down the tubes at 8,000 x g for 15 s to settle the beads at the bottom, remove frothing, and transfer the supernatant completely from the tube to a 1.5 mL microcentrifuge tube. Some beads may also get transferred during this step.</li>
	<li>Centrifuge the supernatant at 15,000 x g for 1 min.</li>
	<li>Transfer the supernatant to a clean 2 mL microcentrifuge tube. The supernatant may still contain some suspended particles.</li>
	<li>Add 200 &#181;L of precipitant solution and vortex at maximum speed for 5 s. Incubate at 4 &#176;C for 5 min. 
	NOTE:&#160;Incubation at 4 &#176;C increases the efficiency of precipitation of proteins and cellular debris while DNA remains in solution. It is possible to pause the protocol at this step for up to 2 h without a significant decrease in the yield and quality of the extracted DNA.</li>
	<li>Centrifuge at 15,000 x g for 1 min at room temperature (RT). Avoid the pellet and transfer the clear supernatant to a clean 2 mL microcentrifuge tube.</li>
	<li>Add 600 &#181;L of binding buffer per 700 &#181;L of supernatant and vortex at maximum speed for 5 s.</li>
	<li>Load 700 &#181;L of the lysate onto a silica spin column, incubate for 2 min, and centrifuge at 15,000 x g for 1 min. 
	NOTE: Incubation of lysate on silica column enhances the binding of DNA to the column, resulting in better DNA yield.</li>
	<li>Discard the flow-through and repeat step 2.14 until all the lysate has passed through the spin column.</li>
	<li>Carefully place the spin column into a clean 2 mL collection tube. Ensure that no flow-through is splashed onto the spin column.</li>
	<li>Add 500 &#181;L of wash buffer to the spin column and centrifuge the spin column at 15,000 x g for 1 min.</li>
	<li>Discard the flow-through and return the spin column to the same 2 mL collection tube.</li>
	<li>Add 500 &#181;L of ethanol-wash buffer to the spin column. Centrifuge at 15,000 x g for 1 min.</li>
	<li>Discard the flow-through and place the spin column into a new 2 mL collection tube.</li>
	<li>Centrifuge at 16,000 x g for 2 min to remove residual ethanol. Place the spin column into a new 1.5 mL tube.</li>
	<li>Add 100 &#181;L of elution buffer (pre-heated to 55 &#176;C) to the center of the white filter membrane and incubate for 5 min. 
	NOTE: Heated elution buffer, along with incubation on the membrane, increases the efficiency of DNA elution, especially high-molecular-weight DNA.</li>
	<li>Centrifuge at 15,000 x g for 1 min. Discard the spin column.</li>
	<li>To the eluted DNA, add 10 &#181;L of 3 M NaCl (1/10 volume of DNA eluate) and 250 &#181;L of chilled absolute ethanol (2.5 volume of DNA eluate). Invert to mix well and spin down the contents at 8,000 x g for 15 s.</li>
	<li>Incubate the DNA-ethanol mixture at -20 &#176;C for at least 1 h. 
	NOTE: The incubation of the DNA-ethanol mixture can be increased to 16 h without a significant decrease in the yield or quality of the extracted DNA.</li>
	<li>Centrifuge at 19,000 x g at 4 &#176;C for 30 min. Discard the supernatant by decantation.</li>
	<li>Add 500 &#181;L of chilled 70% ethanol to the pellet.</li>
	<li>Centrifuge at 19,000 x g at 4 &#176;C for 15 min. Discard the supernatant by decantation.</li>
	<li>Invert and gently tap the tube on tissue paper to remove residual ethanol by capillary action.</li>
	<li>Dry the DNA pellet completely by keeping the tubes open on a heating block for 5-10 min at 37 &#176;C till all ethanol has evaporated.</li>
	<li>Re-suspend the DNA pellet in 30-50 &#181;L of autoclaved double distilled water (ddH2O). Incubate at 37 &#176;C for 5-10 min.</li>
	<li>Spin down the DNA at 8,000 x g for 15 s.</li>
	<li>Select the dsDNA application under the Nucleic Acids settings in a Nanodrop spectrophotometer.
	<ol>
		<li>Clean the pedestal with lint-free tissue paper and water. Use ddH2O to blank the instrument and then load 2 &#181;L of the DNA sample on the pedestal.</li>
		<li>Measure the concentration and absorbance ratios (A260/280 and A260/230) to determine the purity.</li>
	</ol>
	</li>
	<li>Store the DNA sample at -20 &#176;C.</li>
</ol>
3.&#160;Precipitation of polymerase chain reaction (PCR)-amplified linear DNA to check DNA recovery across a range of molecular weights
<ol>
	<li>Using genomic DNA from E. coli MG155, amplify gene fragments by PCR to generate a pool of linear fragments from the following genes: fusA, lacZ, gapA, rpsD, 16S rRNA, marR (see Table 2 for primer sequences and PCR conditions).</li>
	<li>Purify the PCR products using the PCR purification kit and pool the PCR products together.</li>
	<li>Divide the pooled amplicons into 1.5 mL microcentrifuge tubes with equal volumes- label one tube as &#39;Input DNA&#39;.</li>
	<li>Add PEG and NaCl according to the desired conditions (9%, 20%, 30% PEG-8000; 0.3 M, 1.2 M NaCl) to each of the tubes except the one labeled &#39;Input DNA&#39; and make up the final volume to 1 mL.</li>
	<li>Incubate the tubes overnight (~16-18 h) at 4 &#176;C. 
	NOTE: For the rest of the steps till 3.16, &#39;Input DNA&#39; is kept unchanged in the fridge.</li>
	<li>Spin the tubes in a table-top microcentrifuge according to the desired speed (15,000 x g or 20,000 x g) for 1 h at 4 &#176;C.</li>
	<li>Carefully remove 900 &#181;L of the supernatant from the side opposite the pellet using a micropipette.</li>
	<li>First ethanol wash: Add 900 &#181;L of chilled 70% ethanol to the pellet and mix by gently inverting the tubes.</li>
	<li>Spin the tubes at the same speed as used in step 3.6 (15,000 x g or 20,000 x g) for 30 min at 4 &#176;C.</li>
	<li>Remove the supernatant by decantation.</li>
	<li>Second ethanol wash: Add 500 &#181;L of chilled 70% ethanol to the pellet.</li>
	<li>Spin the tubes at the same speed as used in step 3.6 (15,000 x g or 20,000 x g) for 30 min at 4&#176;C. Discard the supernatant by decantation.</li>
	<li>Invert and gently tap the tube on tissue paper to remove residual ethanol by capillary action.</li>
	<li>Dry the DNA pellet completely by keeping the tubes open on a heating block for 5-10 min at 37 &#176;C until all ethanol evaporates.</li>
	<li>Re-suspend the pellet in 20 &#181;L of autoclaved double-distilled water.</li>
	<li>Measure the concentration of DNA precipitated by the different conditions and the &#39;Input DNA&#39; using a Nanodrop spectrophotometer or fluorometer.</li>
	<li>Load the precipitated DNA on a 1% agarose gel for DNA visualization.</li>
</ol>
&#160;&#160;

Efficient Technique for Assessing Environmental AMR by Enriching Low-MW DNA

The authors declare no conflicts of interest.

AMR is one of the top 10 health threats today, as listed by the WHO, and environmental surveillance for AMR is recognized as an important tool across the world. As mentioned in the introduction, a comprehensive record of environmental AMR includes low-molecular-weight, fragmented, and extracellular DNA. The pre-processing protocol reported here using a high concentration of PEG combined with salt (30% PEG and 1.2 M NaCl) achieves this result by enriching the proportion of low-molecular-weight DNA without impacting extrac...

SARS-CoV-2 and its aftermath underlined the importance of environmental surveillance in monitoring and predicting infectious disease outbreaks1,2. While viral pandemics are apparent, the rise of antimicrobial resistance (AMR) is often described as an insidious pandemic and one that constitutes a leading public health concern across the world3,4. Consequently, there is an urgent need for coordinated strategies to understand the evolution and spread of AMR. Water bodies, as well as wastewater, can serve as reservoirs for both pathogens and AMR5,6,7,8. Shared water sources are, therefore, a potent source of disease transmission among humans, particularly in low and middle-income countries (LMIC) where poor hygiene and over-population go hand in hand9,10,11. Testing of water sources has long been employed to assess community health12,13,14. Recently, wastewater from urban sewage treatment plants proved a good advance indicator of COVID cases in the clinic1,2,15,16,17,18.
Compared with monitoring specific diseases, detecting and tracking AMR in the environment poses a more complex problem. The large number of antibiotics in use, diverse resistance genes, different local selection pressures, and horizontal gene transfer among bacteria make it difficult to assess true AMR burden and, once assessed, to correlate it with clinical observations19,20,21,22. As a result, while concerted surveillance of clinical AMR is being carried out by several organizations across the world3,23,24, environmental AMR monitoring is still in its infancy, reviewed in19,25,26.
In recent years, different methods for tracking environmental AMR have been reported5,27, reviewed in28,29. The starting point of most of these is the extraction of good quality DNA from heterogenous environmental samples, in itself a challenge. Additionally, environmental DNA is typically fragmented because of exposure to hostile surroundings. Fragmented extracellular DNA has long been recognized as an important reservoir of AMR genes (reviewed in30,31,32), with the added potential to enter and leave bacteria via horizontal gene transfer. Hence, it is important that any protocol that aims to measure AMR burden in the environment should sample linear and low-molecular-weight DNA as best as possible. Surprisingly, there has been little focus on developing methods specific to high-yield extraction of linear and low-molecular-weight DNA: this work focuses on addressing the gap.
A common and simple method to precipitate DNA is to combine polyethylene glycol (PEG) and salts such as sodium chloride (NaCl)33. PEG is a macromolecular crowding agent used to achieve size-specific precipitation of DNA fragments34,35. The lower the PEG concentration, the higher the molecular weight of DNA that can be efficiently precipitated. Many studies have used PEG during environmental extraction of DNA and RNA1,2 (summarized in Table 117,33,36,37,38,39) either in the final step 33,36,37or to concentrate large water samples for extraction of viral particles as with SARS-CoV-215,40. In the current work, it is found that the PEG concentrations used previously for environmental DNA extractions (largely determined by viral surveillance protocols) do not capture low-molecular weight linear DNA. Therefore, they lose out on sampling short DNA fragments and are unsuitable for assessing AMR content accurately. This study has exploited the properties of polyethylene glycol and sodium chloride to effectively precipitate low-molecular weight linear DNA fragments at a high yield that can, in the future, lead to a cost-effective DNA extraction method. This method can be used to enrich the proportion of fragmented and low-molecular-weight DNA from complex natural samples, thus capturing a more accurate picture of environmental AMR. With a little further refinement, the technique lends itself to easy and low-cost application by local municipal corporations and other government bodies to use as a surveillance tool with minimal technical training.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-seat microcentrifuge</td>
			<td>Eppendorf Centrifuge 5425 R</td>
			<td>EP5406000046</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol (Emsure ACS, ISO, Reag. Ph Eur Ethanol absolute for analysis)</td>
			<td>Supelco</td>
			<td>100983-0511</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top refrigerated centrifuge</td>
			<td>Eppendorf Centrifuge 5920 R</td>
			<td>EP5948000131</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>BioRad</td>
			<td>12003153</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy PowerSoil Pro Kit</td>
			<td>Qiagen</td>
			<td>47014</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy PowerWater Pro Kit</td>
			<td>Qiagen</td>
			<td>14900-100-NF</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (dNTP Mix 10mM Each,0.2 mL, R0191)</td>
			<td>Thermo Fisher</td>
			<td>R0191</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq DNA Polymerase, 5 U/&#181;L + 10x DreamTaq Buffer*</td>
			<td>Thermofscientific</td>
			<td>EP0702</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Gel 1 Kb Plus Express DNA Ladder</td>
			<td>Invitrogen</td>
			<td>10488091</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiamp PCR tubes 0.2 mL</td>
			<td>Tarsons</td>
			<td>510051</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water for PCR</td>
			<td>HiMedia</td>
			<td>ML065-1.5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop OneC Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>13400519</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>SRL</td>
			<td>54866</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR &#38; Gel Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28506</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer (with WiFi)</td>
			<td>Thermofisher</td>
			<td>Q33238</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Thermofisher</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubitt reagent kit for ds DNA, broad range</td>
			<td>Thermo Scientific</td>
			<td>Q32853 (500 assays)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>HiMedia</td>
			<td>TC046M-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Cycler (ProFlex 3 x 32-well PCR System)</td>
			<td>Applied Biosystems</td>
			<td>4484073</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard Genomic DNA Purification Kit</td>
			<td>Promega</td>
			<td>A1125</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhanced extraction of low-molecular weight dna from wastewater for comprehensive assessment of antimicrobial resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

Establishment of a protocol for high-yield extraction of DNA from wastewater samples 
A modified version of previously established protocols was used for the extraction of high-quality DNA and RNA from water samples17. The samples were sourced from open drains as well as sewage treatment plants in the Delhi-NCR region of North India. After pre-processing using PEG and NaCl (Figure 1), the samples were processed th...

Author Spotlight: Understanding and Detecting Environmental Antimicrobial Resistance by Combining Culture-Based Techniques and Genomics

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

enhanced-extraction-low-molecular-weight-dna-from-wastewater-for

Research

JoVE Journal

Biology

877 Views.  Ashoka University. Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

Video: Author Spotlight: Understanding and Detecting Environmental Antimicrobial Resistance by Combining Culture-Based Techniques and Genomics

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 ...

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.

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 ...

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 ...

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 ...

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 ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...