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Learn more about DNA Extraction

DNA Extraction
Kelly M. Elkins, in Forensic DNA Biology, 2013
Background
Deoxyribonucleic acid (DNA) extraction is the process by which DNA is separated from proteins,
membranes, and other cellular material contained in the cell from which it is recovered. This
extraction can be one of the most labor-intensive parts of DNA analysis. Extraction methods may
require an overnight incubation, may be a protocol that can be completed in minutes or a couple of
hours, or may be a recent procedure that employs reagents for which this step can be skipped
completely.
The DNA extraction process requires careful handling of biological material to prevent sample
contamination and crossover. Tubes should be carefully labeled, especially when transfers are
required. Robots may be employed to extract reference samples and some evidence samples, but
other evidentiary samples may require the direct attention of a DNA analyst.
The simplest cells, such as bacteria cells, are prokaryotes. These prokaryotes comprise a lipid
bilayer outer membrane and a cytoplasm containing a circular chromosome, proteins inorganic
salts and metal ions, sugar molecules, and other elements of cell machinery. Humans, animals, and
plants are composed of eukaryotic cells; these cells also have a lipid bilayer outer membrane and
cytoplasm containing proteins, sugars, lipids, and inorganic ions of various types and function.
However, eukaryotic cells also contain other membrane-enclosed compartments called organelles.
The nucleus of a cell is an organelle that houses 46 chromosomes, and the mitochondria each
house a circular DNA chromosome, all of which direct the production of proteins. The
mitochondrial chromosome is 16,569 bp. The mitochondrial proteins are used as some of the
metabolic machinery for digestion of sugars and fats and production of most of the energy for the
cell. Other organelles are involved in the synthesis and modification of proteins, sugars, and fats,
and other molecules used by the cell or in its signaling activities. The genes that code for heritable
traits—including height, blood type, hair color, eye color, skin color, and temperament—are found
on chromosomes in the nucleus of the eukaryotic cell. In plants, the additional chloroplast
chromosome contains genes for photosynthesis. Forensic scientists are largely unconcerned with
the genes that code for proteins or regulatory elements of the DNA; however, forensic scientists
are interested in isolating DNA from the nuclear, mitochondria and chloroplast (if present)
chromosomes to evaluate the sequences, base and repeat polymorphisms, including SNPs and
STRs, respectively, that have proved useful for linking a suspect to a crime scene.
DNA is highly negatively charged because of its phosphate groups. It is stabilized by magnesium
in the cell when unwound. Nuclear DNA consists of 3.2 billion bases in humans. It is organized
into chromosomes in part by coiling around positively charged proteins called histones to form
nucleosomes. Magnesium is also integral to the function of proteases, enzyme proteins that cut up
DNA.
Because of the lipid structure of the cell (and nuclear) membrane(s), presence of proteases and
magnesium, and coiling of DNA around histones, many of the available DNA extraction
procedures have common elements. Indeed, the extraction of DNA generally follows three basic
steps:
1.
Lyse (break open) the cells.
2.
Separate the DNA from the other cell components.
3.
Isolate the DNA.
The cell membrane is disrupted by any of the following methods: using heat to increase fluidity,
dithiothreitol (DTT) to reduce disulfide bonds, or a detergent, such as sodium dodecyl sulfate
(SDS), to disrupt the membrane. Proteins, including nucleases, are inactivated by heat
denaturation or by digestive enzymes, including proteinase K, to cut them up. The temperature
must be kept below 60° C and the period must be kept sufficiently short (15 to 20 minutes) if
nondegraded, high-molecular-weight DNA is required. Either the magnesium needed for nuclease
activity or DNA is immobilized on a solid phase and eluted by buffer/salt. If the DNA remains in
the aqueous phase, it is separated from the other cellular materials including proteins and lipids by
centrifuging the latter to the bottom of the tube or partitioning them in organic solvents.
There are four commonly used extraction procedures for DNA extraction (Hoff-Olsen et al.,
1999):
1.
Organic (variations of phenol/chloroform): use of a multistep liquid chemical process that is labor
intensive but produces a high yield and very-clean double-stranded extracted DNA sample
2.
Inorganic Chelex or silica methods: simple and cheap one-tube extraction process in which Mg2+
binds to resin beads and yields a single-stranded DNA product
3.
Solid phase extraction methods (e.g., Promega’s DNA IQ (Eminovic et al., 2005, DNA IQ
manual), Applied Biosystems’ PrepFiler (Brevnov et al., 2009, PrepFiler manual), and Qiagen’s
QIAamp kits (Castella et al., 2006, Greenspoon et al., 1998)): simple extraction process in which
the DNA binds to paramagnetic or silica beads
4.
Differential extraction: a multistep process used to separate sperm from other cells using DTT;
used for analyzing biological evidence from sexual assault cases (Drobnic, 2003)
For organic extraction, the cells must be lysed using gentle heat to disrupt the membranes with the
assistance of SDS and DTT. Proteinase K must be added to digest the proteins, including
nucleases. In the second step of the PCIA extraction, the DNA must be separated from the other
cellular components using the phenol-chloroform-isoamyl alcohol (PCIA) reagent. When the
PCIA is added, two phases appear: the organic (bottom) and the aqueous (DNA-containing) phase
(top) after centrifugation; the DNA-containing portion is removed by pipetting off the top layer.
DNA is more soluble in the aqueous state compared to the organic phases. It is important to
remove all of the organic extraction agents because phenol will degrade DNA. Phenol has a
density of approximately 1 g/mL and can easily invert with the aqueous phase. Lastly, in the third
step, the DNA can be filtered and concentrated by centrifugation through a Microcon, Centricon,
or Vivacon 100 filter in which the DNA is captured on the membrane while all other aqueous
components including protein fragments, residual organics, and potential PCR inhibitors are
allowed to pass through the membrane (100 kilodalton cut-off) (Moore, 1998). The DNA is eluted
by inverting the filter and centrifuging using a desired volume of elution buffer or nuclease-free
water (20 to 50 μL). Using the filter, the DNA is concentrated in a small volume for subsequent
amplification steps. This method yields a double-stranded DNA extract and the highest yield but
requires multiple tube changes which increase the possibility of contamination error.
The basic Chelex procedure (Walsh et al., 1991) consists of an overnight incubation to disrupt the
cell membrane and allow the 5% Chelex solution (styrene divinylbenzene copolymers containing
paired iminodiacetate ions), which has a high affinity for and binds polyvalent metal ions such as
magnesium, and Proteinase K to digest the nucleases, and boiling the sample in a 5% Chelex
solution to burst the remaining membranes, denature the proteins and denature the DNA in the
cells. The result is a denatured sample of single-stranded DNA that remains in the supernatant
after centrifugation. The single-stranded DNA is then ready to be concentrated, and if necessary it
can be quantified and amplified. Because this is a single-tube reaction, there is less potential for
contamination and mis-pipetting. Silica and glass beads can be substituted for Chelex in this type
of extraction procedure (Dederich et al., 2002).
The DNA IQ System by Promega employs silica-coated paramagnetic beads, which bind the DNA
(up to 100 ng sample). The magnetic beads are drawn to the bottom of the tube using a magnetic
holder, and the other cellular components are washed away. The process may be automated using
robotic systems and is ideal for consistent-concentration samples including paternity and reference
samples. The kit contains the proprietary resin and specialized lysis, elution, and wash buffers for
the procedure (Eminovic, et al., 2005, DNA IQ manual).
The Qiagen QIAamp DNA Micro Kit employs silica-based bead extraction method using a spin
column. The silica beads bind the DNA under high chaotropic salt conditions. The kit contains the
spin columns and sample and lysis buffer, none of which contain hazardous chemicals. The cell
lysate is combined with alcohol and placed into the spin column, which is inserted into a tube.
Proteins and divalent cations, positively charged ions, including magnesium, are removed using
multiple buffer washes (specific formulations are available for different cell/sample types) and
centrifugation steps. Pure DNA is eluted from the membrane using the desired quantity of sterile
water or Tris-EDTA buffer. The procedure can be automated (Castella et al., 2006, Greenspoon et
al., 1998).
FTA filter paper cards use technology licensed by Whatman from Flinders University. The
technique was originally developed by Burgoyne and Fowler at Flinders University in Australia in
the 1980s. The nucleic acid is protected from degradation by nucleases from reagents contained in
the filter paper including a weak base, a chelating agent, an anionic surfactant or detergent, and
uric acid (or a urate salt) to a cellulose-based matrix (filter paper). A sample containing DNA can
then be applied to the treated filter paper for preservation and long-term storage. The cards are
widely used because they are small, can be stored at room temperature, and ship easily (Thacker et
al., 2000).
In this experiment DNA will be extracted from questioned and known samples containing
biological material using one or more of the following methods in one to two sessions.
Read full chapter
DNA Extraction Methods
John M. Butler, in Advanced Topics in Forensic DNA Typing: Methodology, 2012
DNA extraction involves separating the nucleic acids in a cell away from proteins and other
cellular materials. Different methodologies widely used by forensic DNA scientists include
organic, Chelex, or solid-phase extraction. Post-extraction filtration is sometimes used to
concentrate low amounts of recovered DNA sample. A differential extraction that exploits
chemical differences in sperm cell coatings can be used with sexual assault evidence to separate
sperm from epithelial cells. Laser-capture microdissection technologies now enable physical
separation of cells through selective recovery of individual sperm or other cells. It is important
with any DNA extraction technique to remove as many substances as possible that could interfere
with downstream testing and cause the extracted DNA molecules to break down over time.
Although the addition of buffer components that can overcome inhibition, direct PCR now permits
by-passing the DNA extraction and quantitation steps.