EXTRACTION OF DNA FOR

ANALYSIS
DNA is present in every nucleated cell and is therefore present in biological materials
left at crime scenes. DNA has been successfully isolated and analyzed from a variety of
biological materials. Introduction of the polymerase chain reaction (PCR) has extended
the range of possible DNA samples that can be successfully analyzed because many
copies are made of the DNA markers to be examined. The different types of biological
evidence can be used to associate or to exclude an individual from involvement with a
crime. In particular, the direct transfer of DNA from one individual to another individual
meaningful and legally accepted in court. DNA testing techniques have become so
sensitive that biological evidence too small to be easily seen with the naked eye can be
used to link suspects to crime scenes. The evidence must be carefully collected,
preserved, stored, and transported prior to any analysis conducted in a forensic DNA
laboratory.
DNA EXTRACTION
A biological sample obtained from a crime scene in the form of a blood or semen stain or
a liquid blood sample from a suspect or a paternity case contains a number of substances
besides DNA. DNA molecules must be separated from other cellular material before they
can be examined. Cellular proteins that package and protect DNA in the environment of
the cell can inhibit the ability to analyze the DNA. Therefore, DNA extraction methods
have been developed to separate proteins and other cellular materials from the DNA
molecules. In addition, the quantity and quality of DNA often need to be measured prior
to proceeding further with analytical procedures to ensure optimal results.
The most common DNA extraction protocols include basic components discussed below.

1. CELL AND TISSUE DISRUPTION - In most DNA extraction protocols, enzymatic

digestions, such as those with proteinase K, are used for cell and tissue disruption. The
disruption process can also be carried out by boiling and by using alkali treatment and
mechanical methods. Materials such as bone and teeth can be frozen in liquid nitrogen and
then ground to a fine powder with a mortar. In such specimens, cells containing DNA are
embedded in a calcified matrix. A decalcification process removes calcium ions from the
matrix, thus making the specimen suitable for DNA extraction. The most commonly used
decalcifying agents for DNA extraction purposes are chelating agents such as
ethylenediaminetetraacetic acid (EDTA).

2. LYSIS OF CELLULAR AND ORGANELLE MEMBRANE-During or after tissue disruption,

membranes—including those of cells, nuclei, and mitochondria are lysed in order to release
DNAs, including nuclear and mitochondrial DNA.
• The lysis can be carried out using salts such as guanidinium salts and detergents such as
sarkosyl and sodium dodecyl sulfate (SDS). These substances can destroy membranes,
denature proteins, and dissociate proteins such as histones from DNA.
The lysis procedure is usually carried out in a buffer, such as Tris, in order to maintain a
pH where endogenous deoxyribonucleases (Dnases) remain inactive. Chelating agents
such as EDTA can therefore be used to inhibit Dnase activities.
• Furthermore, reducing agents such as mercaptoethanol or dithiothreitol (DTT) can be
used to inhibit the oxidization processes that can damage DNA.

3.REMOVAL OF PROTEINS AND CYTOPLASMIC SUBSTANCES

After lysis, cytoplasmic constituents, such as proteins and liquids that interfere with DNA
extraction, are removed. Proteins and lipids are usually removed by one or more rounds of
extraction with organic solvents such as phenol–chloroform mixtures. Another strategy to
remove cytoplasmic constituents is to utilize the reversible binding of DNA to a solid
material such as silica, which selectively binds DNA in chaotropic salt solutions. The
proteins and cytoplasmic constituents can then be removed through washing steps.
4.STORAGE OF DNA SOLUTIONS

Purified high-molecular-weight DNA is usually stored in TE buffer (10 mM

Tris-HCl, 1 mM EDTA, pH 8.0). EDTA is usually included in storage solutions
to chelate divalent cations and thereby inhibit Dnases. Such a DNA solution
may be stored at 4°C or −20°C. For long-term storage, −80°C is recommended.
Methods of DNA Extraction
• 1 Extraction with Phenol–Chloroform
This method is also called organic extraction. Major steps include the following:
1 Cell Lysis and Protein Digestion
These steps can be achieved by digestion with proteolytic enzymes such as proteinase K before
extraction with organic solvents.
2 Extraction with Organic Solvents
The removal of proteins is carried out by extracting aqueous solutions containing DNA with a
mixture of phenol:chloroform:isoamyl alcohol. Phenol is used to extract the proteins from the
aqueous solution. Although phenol has a slightly higher density than water, it is sometimes
difficult to separate it from the aqueous phase. Therefore, chloroform is utilized as it has a
higher density than phenol. As a result, the phenol–chloroform mixture forms the organic phase
at the bottom of the tube and is easily separated from the aqueous phase. Isoamyl alcohol is
often added to the phenol–chloroform mixture to reduce foaming. During partition, DNA is
solubilized in the aqueous phase, while lipids are solubilized in the organic phase. Proteins are
located at the interface between the two phases.
3 Concentrating DNA
Two common methods for concentrating DNA are ethanol precipitation and
ultrafiltration. In the first method, the DNA is precipitated from the aqueous
solution with ethanol and salts. Ethanol depletes the hydration shell of DNA, thus
exposing its negatively charged phosphate groups. The precipitation can only occur
if sufficient quantities of cations are present in the solution. The most commonly
used cations, such as ammonium, lithium, and sodium, neutralize the charges on
the phosphate residues of DNA, forming a precipitate. Ultrafiltration is an
alternative to ethanol precipitation for concentrating DNA solutions.
ADVANTAGE –Organic extraction method works well for recovery of high molecular
weight DNA
DISADVANTAGES-It is time-consuming, involves the use of hazardous chemicals, and
requires the sample to be transferred between multiple tubes(contamination).
2.Extraction by Boiling Lysis and Chelation
This technique, also called the Chelex® extraction method. It usually includes the following
steps:
1 Washing
This step removes contaminants and inhibitors that may interfere with DNA amplification.
For example, heme compounds found in blood samples
2 Boiling
Cells are suspended in a solution and incubated at 56°C, where Dnases are not active, for
20 min. This preboiling step softens cell membranes and separates clumps of cells from
each other. The cells are then lysed by heating to boiling temperature in order to break open
the membranes and to release the DNA. Additionally, the lysis of cells releases all of their
cellular constituents, including Dnases. The Dnase degradation of the extracted DNA can be
blocked by applying a chelating resin (Chelex® 100) during the extraction process.
Chelex® 100 is an ion-exchange resin composed of styrene divinylbenzene copolymers.
The paired iminodiacetate ion groups in Chelex® 100 act as chelators by binding to divalent
metal ions such as magnesium. Magnesium is a cofactor of endogenous Dnases. Thus,
sequestering magnesium in the solution using Chelex®100 protects DNA from degradation
by Dnases.
3 Centrifugation
Brief centrifugation is performed to pull the Chelex® 100 resin and cellular debris to the
bottom of the tube. The supernatant is used for DNA analysis.

ADVANTAGES- This method is simple and rapid and uses only a single tube for extraction,
thus reducing the risks of contamination and sample mix-ups.
DISADVANTAGES- However, the heating step of this method disrupts and denatures
proteins and also affects the chromosomal DNA. The resulting DNA extracted from the
solution is fragmented single-stranded DNA. Thus, the DNA extracted is not suitable for
RFLP analysis because RFLP requires double-stranded DNA samples. The DNA obtained by
lysis and chelation can only be used for PCR-based DNA analysis.
3.Silica-Based Extraction

The method of adsorbing DNA molecules to solid silica surfaces is used for extraction . The
method is based on the phenomenon that DNA is reversibly adsorbed to silica - silicon dioxide
(SiO2)—in the presence of high concentrations of chaotropic salts. These salts are used to
denature proteins. Additionally, chaotropic salts can facilitate the adsorption of DNA to silica.
Common chaotropic salts utilized for DNA extraction include guanidinium salts such as
guanidinium thiocyanate (GuSCN) and guanidinium hydrochloride (GuHCl).This technique
usually includes the following steps:
1 Cell Lysis and Protein Digestion
This is carried out by proteinase K digestion. The cell membranes are broken open, and DNA
is released.
2 DNA Adsorption onto Silica
This step utilizes silica as the stationary phase in a membrane configuration to which the DNA
in the cell lysate binds. Adsorption of the DNA to the silica occurs in the presence of high
concentrations of chaotropic agents . The adsorbed DNA is largely double stranded.
3 Washing
This step removes chaotropic agents and other contaminants. An ethanol-based wash solution
is used. This wash solution does not remove DNA from the silica. The chaotropic agents and
contaminants that are present in the solution can be removed using the ethanol-based wash
solution.
4 Elution of DNA
The adsorbed DNA can be eluted by rehydration with aqueous low-salt solutions. The eluted
DNA is double stranded and can be used for a wide variety of applications.
ADVANTAGES- Silica-based extraction methods yield high-quality DNA. Silica membrane
devices can also be adapted for automation; for example, this can be done by using 96-well
silica membrane plates and a variety of robotic platforms. Another type of device utilizes
silica-coated paramagnetic particles that adsorb DNA in the solution . A magnet is used for
particle capture instead of centrifugation or vacuum filtration. The magnetic particles can be
resuspended during the wash steps, and the solution containing contaminants and cellular
materials is then discarded. DNA is eluted after washing. This device can also be adapted to
automated, high-throughput methods. Over the years, high throughput silica-based procedures
have been developed to process large numbers of samples in parallel. Some of these methods
are adapted for automated DNA extraction platforms
4. DIFFERENTIAL EXTRACTION
This method is very useful for the extraction of DNA from biological evidence derived from
sexual assault cases, such as vaginal swabs and bodily fluid stains. These types of evidence
often contain mixtures of spermatozoa from a male contributor and nonsperm cells such as
epithelial cells from a female victim. Mixtures of individual DNA profiles can complicate data
interpretation. This method selectively lyses the nonsperm and spermatozoa in separate steps
based on the differences in cell-membrane properties of spermatozoa and other types of cells.
Thus, the DNA from spermatozoa and nonsperm cell fractions can be sequentially isolated.
 First, the differential extraction procedure involves preferentially lysing the nonsperm cells
with proteolytic degradation using proteinase. Sperm plasma membrane contains proteins
cross-linked by disulfide bonds. The membrane exhibits a much higher mechanical stability
than nonsperm cells and is thus resistant to proteolytic degradation. The nonsperm DNA is
released into the supernatant and the liquid containing it (the nonsperm fraction) is extracted,
yielding a fraction that predominantly contains DNA from nonsperm cells. To lyse the sperm
cells, it is necessary to cleave the disulfide bonds in addition to proteolytic digestion. The
application of DTT, a reducing agent, is an approach that can be used for cleavage. In the
presence of DTT and proteinase K, the sperm plasma membrane is then lysed . Subsequently,
DNA from the sperm cells can be extracted
ADVANTAGES- This process isolates sperm and nonsperm cell DNA separately for
obtaining DNA profiles from male and female contributors, respectively.

DISADVANTAGES- However, the non-sperm-cell DNA and spermcell DNA may not be
completely separated from one another; this can happen, for example, if the sperm cells have
already lysed due to poor sample conditions. Some sperm DNA may be present in the non-
sperm-cell fraction. Additionally, if a mixture has an abundance of nonsperm cells and fewer
sperm cells, non-sperm-cell DNA may be detected in the sperm fraction. Thus, new methods
that can overcome these problems are highly desired.
5.FTA PAPER

Another approach to DNA extraction involves the use of FTA paper. FTA paper is an
absorbent cellulose-based paper that contains four chemical substances to protect DNA
molecules from nuclease degradation and preserve the paper from bacterial growth. As a
result, DNA on FTA paper is stable at room temperature over a period of several years.
 Use of FTA paper simply involves adding a spot of blood to the paper and allowing the
stain to dry. The cells are lysed upon contact with the paper and DNA from the white
blood cells is immobilized within the matrix of the paper.
 A small punch of the paper is removed from the FTA card bloodstain and placed into a
tube for washing. The bound DNA can then be purified by washing it with a solvent to
remove heme and other inhibitors of the PCR reaction. This purification of the paper
punch can be seen visually because as the paper is washed, the red color is removed with
the supernatant.
 The clean punch is then added directly to the PCR reaction.
ADVANTAGES- A major advantage of FTA paper is that consistent results may be
obtained without quantification. Furthermore the procedure may be automated on a
robotic workstation . For situations where multiple assays need to be run on the same
sample, a bloodstained punch may be reused for sequential DNA amplifications and
typing .
DISADVANTAGE- Unfortunately, dry paper punches do not like to stay in their
assigned tubes and due to static electricity can ‘jump’ between wells in a sample tray.
Thus, this method is not as widely used today as was once envisioned.

However, due to its preservation and storage capabilities, efforts have been made to use
FTA cards for more widespread collection of crime scene evidence .