Isolation of Genomic DNA from Mammalian Cells and Fixed Tissue

Examination of DNA variation is central to understanding the function of mammalian cells, tissues, and whole bodies. Extraction of high‐quality DNA from cells and tissues is necessary for innumerable different experiments. We present protocols for the extraction of DNA from both fresh samples and formalin‐fixed tissue. Methods for extracting DNA have been standardized and streamlined over the past couple of decades and many extraction kits are available for a reasonable cost. In addition, many of the extraction procedures can also be automated for even higher throughput sample preparation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.


INTRODUCTION
Obtaining DNA for genetic studies has become an essential component of virtually all biomedical research. This unit provides an update for simple, cost-effective preparations of DNA from whole blood, human tissue, cultured cells, and saliva/buccal swab samples. When working with fresh or frozen samples, high-molecular-weight DNA suitable for downstream assays, such as genotyping arrays or next generation sequencing, can be routinely isolated. DNA can also be isolated from formalin-fixed, paraffin-embedded (FFPE) tissues, though it is more challenging and there are limitations in the downstream applications for which this DNA is suitable. Over the last couple of decades, the availability and decreased cost of commercial kits has made their use prominent in the field. There are many vendors offering similar types of extraction kits which are a cost-effective option for obtaining high quality DNA from human specimens. Most commercially available extraction methods no longer require the use of toxic chemicals, such as phenol and chloroform, also making them a safer option for researchers. Commercially available kits generally perform as well as manual methods in terms of yield and quality of DNA (Chacon-Cortes & Griffiths, 2014; Guha et al., 2018). Basic Protocol 1 describes the isolation of DNA from fresh or frozen human blood, tissue, or cultured cells. The Alternate Protocol describes how automated machines could be utilized for high throughput sample processing. Basic Protocol 2 describes methods for obtaining and extracting high quality DNA from saliva or buccal swabs collected from inside of the cheek. Basic Protocol 3 outlines how to extract DNA from formalin-fixed paraffin-embedded (FFPE) tissues. For more detailed information about some of the extraction procedures below, a recent book chapter is a useful source of information (Mullegama et al., 2019).
CAUTION: When working with human samples, it is essential to utilize safety precautions for biohazardous samples and bloodborne pathogens. These include proper personal protective equipment, waste disposal, and decontamination procedures. Please consult with your institution's Environmental Health and Safety department to ensure you are complying with all guidelines.
NOTE: All protocols involving animals must be reviewed and approved by the appropriate Animal Care and Use Committee and must follow regulations for the care and use of laboratory animals. Appropriate informed consent is necessary for obtaining and use of human study material.

DNA ISOLATION FROM WHOLE BLOOD, TISSUE, AND CULTURED CELLS
Most commercially available extraction kits can be used to extract DNA from multiple types of human samples, such as blood, tissue, and cultured cells. In addition, there are kits that are designed to handle varying amounts of starting material. The yield of DNA is mainly dictated by the amount and type of starting material used as input for the extraction process. The main components of the extraction kits and their purposes are described. It is crucial to follow the specific instructions that come with each kit. Basic Protocol 1 describes the extraction of DNA from human blood, tissue, or cultured cells using a column-based kit such as the QIAamp DNA Blood Mini Kit or the DNeasy Blood and Tissue Kit. Though far from an exhaustive list, Table 1 provides information about most of the commonly used DNA extraction kits in research laboratories.

Materials
Whole blood, cultured cells, or human tissue (fresh or frozen and thawed) DNA extraction kit containing: Lysis buffer Proteinase K digestion enzyme Wash buffer(s) Elution buffer Water bath Additional reagents and equipment for quantitation of DNA by absorption spectroscopy (Gallagher & Desjardins, 2007) 1. Add a mixture of lysis buffer and proteinase K enzyme to an aliquot of whole blood, cultured cells, or tissue. The lysis buffer contains detergents that will disrupt cellular and nuclear membranes, releasing the cell contents, including the DNA from the nuclei. Incubate the sample at 56°C for a length of time required to digest the proteins in the sample.
The length of the incubation depends on the sample; most human tissue samples will likely require an overnight incubation to achieve complete digestion. Miskimen and Miron resin. The remaining biomolecules and debris will flow through the column when it is centrifuged.
Some kits make use of magnetic beads to capture the DNA. This procedure would require a magnetic plate to bind the beads during the subsequent wash steps.
3. Wash the column with the wash buffers that contain ethanol and various salts to wash away any contaminants or unwanted biomolecules that may have bound to the column.
After the final wash, spin the column for an extra-long spin (e.g., 3 min instead of 1 min) to be sure any residual wash buffer is removed and the ethanol has been dried, as it could interfere with downstream applications.
4. Elute the DNA from the resin in the column by adding an elution buffer in which the DNA is highly soluble. 5. Quantify DNA concentration using UV or fluorescence absorption spectroscopy. Taking a measurement at 260 and 280 nm and comparing the ratio of these numbers (260/280) will give you an indication of the quality of the DNA as well.
DNA can also be run out on an agarose gel at this point to confirm the presence of high molecular weight fragments and minimal degradation.

DNA EXTRACTION USING AUTOMATED MACHINES
Many of the extraction kits and protocols can be modified or run using automated machines. Table 2 lists many of the commonly used automated machines that are

of 12
Current Protocols available for use in research labs. Of note, these machines are generally quite expensive to purchase and require service contracts for preventative maintenance. It might only be a cost-effective choice for labs that routinely extract DNA from hundreds or thousands of samples per year. This Alternate Protocol describes extraction using a QIASymphony machine.

Materials
Whole blood (fresh or frozen and thawed) Reagent cartridge containing all lysis, wash, and elution buffers Pipet tips compatible with extraction machine Tubes for final elution Water bath Additional reagents and equipment for quantitation of DNA by absorption spectroscopy (Gallagher & Desjardins, 2007) 1. Thaw blood quickly in 37°C water bath or aliquot appropriate volume of blood as specified in the machine and kit manual of choice.
Most extractions machines also have kits available for extraction of DNA from cell pellets and tissues as well. These may require some upstream processing to prepare for the lysis step before loading onto the machine.
2. Load the samples, pipet tips, the reagent cartridge, and tubes for elution into the extraction machine.
3. Choose the appropriate extraction protocol for the appropriate sample type and volume, then run the extraction procedure.
As an example, to extract DNA from 24 1-ml aliquots of whole blood, the QIASymphony machine takes about 1.5 hr to run, and other batches can be immediately loaded on the machine as soon as the first run has finished.

Run any clean up or maintenance procedures as necessary on the machine.
It is also important to keep up with preventative maintenance and calibration of the machine as covered by the service contract.
5. Quantify DNA concentration using UV or fluorescence absorption spectroscopy.

DNA ISOLATION FROM SALIVA AND BUCCAL SWABS
Saliva and oral/buccal samples (cheek swabs and brushes) have become increasingly valuable sources of genetic material for clinical applications due to ease of access (noninvasive sample collection), as well as convenient storage and transport procedures that result in adequate DNA yield and quality for genetic studies. Multiple reports indicate that saliva samples provide better quality DNA than buccal samples (Rethmeyer et al., 2013;Rogers et al., 2007). Saliva-derived DNA is now a common alternative to bloodderived DNA, and there are several commercially available saliva collection kits that act to stabilize the samples at ambient temperatures for extended periods prior to DNA extraction. Basic Protocol 2 describes the extraction of DNA from saliva collected using the Oragene-DX Saliva Collection Kit. Table 3 provides information about additional saliva and buccal swab sample collection and DNA extraction kits.
Sample will become turbid as impurities and inhibitors are precipitated.

DNA ISOLATION FROM FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE
There is a potential wealth of information present in tissue samples taken from patients. Almost all tissues biopsied or surgically removed due to a condition or disease involve pathological testing. The most common pathological analysis of tissues involves fixation with formalin followed by embedding the fixed material in a paraffin block. Thin slices of the block can then be made and mounted on slides for staining and examination by a pathologist. Over the last one or 2 decades, there has been a great deal of interest in examining the sequence of the DNA present in these human tissue samples. However, purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue presents several challenges. To purify the DNA, the paraffin must be removed from the sample by volatile chemicals such as xylene. When the tissue is fixed in formalin it creates tangled crosslinked structures of proteins and DNA in the cells of the tissue. It can be difficult to achieve adequate digestion of cellular proteins to purify the DNA. In addition, the resulting DNA that can be isolated consists of short fragments that are not suitable for some applications, such as long-range sequencing. Library preparation techniques have been developed using the polymerase chain reaction (PCR) to amplify short regions of this DNA for use in some next-generation sequencing protocols. Basic Protocol 3 describes the extraction of DNA from FFPE tissue using the QIAamp DSP DNA FFPE Tissue Kit. Table 4 provides information about other common commercially available DNA extraction kits for FFPE tissue. Kresse et al. (2018) compared several FFPE DNA extraction kits and found that most produced comparable sequencing libraries and sequencing data.

CAUTION:
Chemical substances such as xylene should be used in chemical fume hood and proper chemical waste disposal procedures.

Background Information
Since the discovery that DNA is the genetic material for most organisms, there has been much interest in studying the sequence of DNA to better understand biological processes that determine phenotype expression. In order to study DNA structure and function, there first had to be a method to isolate this biomolecule in a relatively pure and intact state. Early methods for DNA isolation were very time-consuming and used harsh chemicals, such as phenol and chloroform. Over the last 2 decades, the methods for DNA extraction have been improved immensely and are now much more streamlined and safer for the person performing the extraction. There has also been the development of automated extraction machines to help facilitate highthroughput isolation of DNA from tens to hundreds of samples simultaneously. In most clinical, research, and forensic laboratories, commercially available kits are being utilized to isolate DNA from human samples, such as blood, tissue, saliva, and even formalinfixed paraffin-embedded (FFPE) tissue that has been collected for pathological analysis. Commercial kits are cost-effective and result in consistent extraction of high-quality DNA for use in many downstream applications such as genotyping or next-generation sequencing.

Critical Parameters and Troubleshooting
The amount and quality of DNA obtained from human samples can vary widely depending on the state and type of the starting material. In general, best laboratory practices would involve minimizing the amount of time between the removal of the bodily fluid or tissue from the human body (or cell culture dish) and storage at a very low temperature (−80°C or liquid nitrogen storage highly recommended). For FFPE tissues, it is critical to optimize the fixation conditions of each tissue type to not over-fix the tissue in the forma-lin. This will make downstream DNA isolation much more difficult and result in lower yield. Most pathology labs have standardized operating procedures for fixing various tissues. When using a kit for DNA extraction, is important to pay attention to the expiration dates of the kits. Many of the wash buffers contain ethanol, which can evaporate and react with water, and result in less-effective washing. Before using DNA in downstream assays, it is highly recommended that the DNA concentration be determined using a fluorometric assay, as this is the most accurate method. In addition, DNA quality should be determined by running DNA on a 1% agarose gel or E-Gel (Invitrogen) to ensure that the DNA is not degraded.

Understanding Results
Again, the amount of DNA can vary depending on the starting material. From whole blood, we can routinely isolate about 50 μg of DNA from 3 ml. Saliva tends to have more variation. We have isolated an average yield of 16 μg per 0.5 ml of saliva with a range of 0.8 to 97 μg. The DNA obtained from fresh/frozen human samples is suitable for most downstream applications including PCR, genotyping, or next-generation sequencing. FFPE tissues have the most variability as anywhere from 1 to 50 μg of DNA may be obtained. The DNA obtained from FFPE tissue would be suitable for PCR and most short-read NGS applications, but not for long-range sequencing.

Time Considerations
The column-based kits for DNA extraction are straightforward. It takes approximately 2 hr of hands-on time to extract 12 wholeblood samples and approximately 3 hr to extract 24 whole-blood samples. For an automated extraction machine, such as the QIASymphony, the setup takes approximately 30 min of hands-on time, then the run takes approximately 1.5 hr for 24 samples. Saliva extraction using the Oragene kit is also straightforward. There are a few incubation steps, so it is approximately 1 hr of hands-on time for 12 samples, then an overnight resuspension of the DNA is recommended. The time constraints for extracting DNA from tissue (fresh or FFPE) depend on the proteinase K digestion step. Often, this is an overnight digestion of approximately 16 hr or more. After that, the column-based extraction steps are straightforward, the same as for whole-blood.