DNA Isolation / Extraction

FTA paper

FTA paper provides a means for collecting, transporting, anchor storing blood
samples and possibly other biological specimens. Blood can be deposited directly
from a finger-prick onto the paper, or blood from an EDTA vacutainer tube can be
deposited by pipette onto the FTA paper. Cell membranes are lysed in the FTA
matrix, and the DNA from the cells becomes entangled in the paper. The filter paper
entraps DNA such that it is essentially immobilized. Thus, proteins and cellular debris
can be washed away from the sample, and yet the DNA remains intact and is purified
within the paper. The washed paper, containing the DNA, is the conduit for sample
analysis in restriction fragment-length polymorphism (RFLP) or PCR-based assays.
The FTA paper is impregnated with denaturants that prevent bacterial and fungal
growth, as well as oxidative and UV damage. Thus, blood and other samples stored
on FTA paper will be stable for long time periods and may be stored at ambient
temperature.

DNA Extraction:

DNA can be extracted from most types of biological material found at a crime scene.
The success of DNA typing relies on the isolation of DNA of sufficient quantity,
quality, and purity. Depending on the typing procedure, the required quantity (i.e., the
amount of retrievable DNA) and quality (i.e., the length of the DNA molecules) of
DNA can vary widely. Purity of the DNA extract refers to a quality of cleanliness,
such that subsequent analytical assays can be carried out effectively. Generally, DNA
extraction protocols that overcome, remove, or dilute enzymatic inhibitors are
desirable. Although many assays are similarly based, there is a tremendous number of
variations, and many can work effectively.

Ideally, extraction protocols should be simple and inexpensive to perform. Procedures

should be suitable for extraction of DNA from small liquid blood samples,
bloodstains, and other tissues. Procedures suitable for extraction of DNA from small
liquid blood samples and bloodstains are based on standard DNA extraction methods.
Standard DNA extraction procedures may entail (i) organic solvent, (ii) salting out
methods, or (iii) cation exchange resins such as Chelex 100 (Bio-Rad Laboratories,
Hercules, CA. USA). Organic and salting out methods are compatible with various
typing procedures, including restriction fragment-length polymorphism (RFLP)
typing and polymerase chain reaction (PCR)-based analyses and are routinely used.

The organic extraction procedure yields highly purified DNA. The process involves
(i) lysis of the red cells with SSC buffer (NaCl, sodium citrate),
(ii) centrifugation,
(iii) lysis of the pelleted white cells, and
(iv) digestion of proteinaceous materials by incubation in a sodium acetate, sodium
dodecyl sulfate (SDS), and proteinase K solution.

DNA released into the solution is extracted with phenol to remove proteinaceous
material, and chloroform is present to remove residual phenol. The DNA is
precipitated from the aqueous layer by the addition of cold ethanol and salt and by
subjecting the solution to centrifugation. Considerable care must be exercised not to
disturb the DNA pellet when removing the ethanol. Residual ethanol may be removed
under vacuum in a specially designed centrifuge. The resultant pellet should be
slightly moist to ensure re-suspension in Tris-EDTA (TE) buffer.

Instead of precipitating the DNA with alcohol, salts can be removed by dialysis. The
simplest way to achieve dialysis is by centrifugation through a filter device, such as
an Amicon® MicroconTM 100 filter unit (Millipore, Bedford, MA, USA). If the
DNA is to be amplified by PCR, as opposed to restriction analysis, a procedure
involving Microcon 100 dialysis of the aqueous layer can be followed.
Sodium dodecyl sulphate (SDS)

Since we are trying to separate many different protein molecules of a variety of

shapes and sizes, we first want to get them to be linear so that the proteins no longer
have any secondary, tertiary or quaternary structure (i.e. we want them to have the
same linear shape). Consider two proteins that are each 500 amino acids long but one
is shaped like a closed umbrella while the other one looks like an open umbrella. If
you tried to run down the street with both of these molecules under your arms, which
one would be more likely to slow you down, even though they weigh exactly the
same? This analogy helps point out that not only the mass but also the shape of an
object will determine how well it can move through and environment. So we need a
way to convert all proteins to the same shape - we use SDS.

SDS is a detergent (soap) that can dissolve hydrophobic molecules but also has a
negative charge (sulphate) attached to it. Therefore, if a cell is incubated with SDS,
the membranes will be dissolved and the proteins will be soluablized by the detergent,
plus all the proteins will be covered with many negative charges. So a protein that
started out like the one shown in the top part of figure 1 will be converted into the one
shown in the bottom part of figure 1. The end result has two important features: 1) all
proteins contain only primary structure and 2) all proteins have a large negative
charge which means they will all migrate towards the positive pole when placed in an
electric field.

Figure 1: This cartoon depicts what happens to a protein (pink line) when it is
incubated with the denaturing detergent SDS. The top portion of the figure shows a
protein with negative and positive charges due to the charged R-groups of the
particular amino acids in the protein. The large H represents hydrophobic domains
where non-polar R-groups have collected in an attempt to get away from the polar
water that surrounds the protein. The bottom portion shows that SDS can break up
hydrophobic areas and coat proteins with many negative charges which overwhelms
any positive charge in the protein due to positively charged R-groups. The resulting
protein has been denatured by SDS (reduced to its primary structure) and as a result
has been linearised.
Non-organic extraction methods that use NaCl or LiCl to salt out protein have been
described. Because salts may not be adequately removed using those procedures,
problems may occur with restriction endonuclease digestion and band shifting
(alteration of DNA mobility). However, with proper care these methods can produce
satisfactory results.

The above procedures are compatible with both RFLP and PCR-based procedures,
because the isolated DNA is double stranded. The Chelex method is only compatible
with PCR-based assays. Basically, Chelex 100 is an ion-exchange resin that binds
cations—removing them from solution— that may inhibit the PCR process. The
Chelex protocol entails placing a small sample in 5% (wt/vol) Chelex and incubating
the sample at 56°C for 30 minutes. After incubation, the sample is boiled for 8
minutes and centrifuged. A portion of the supernatant is used for the PCR. Boiling
denatures the DNA into the single strand format, rendering the DNA unsuitable for
restriction enzyme digestion (i.e., RFLP typing). However, chelex-extracted DNA is
compatible with the PCR. The DNA extract used for the PCR should not contain
residual Chelex; Chelex will bind magnesium ions, which are needed for polymerase
activity.

A method of purification that is particularly useful for reference samples is the

washing away of cellular material from DNA immobilized in FTA paper (Fitzco,
Minneapolis, MN, USA). Blood is spotted onto FFA paper, the cells are lysed, the
DNA released from the cells is immobilized in the FTA matrix, and the sample is
dried. Haem and other cellular debris, which may inhibit enzymatic activity in
subsequent assays, are simply washed away. The washed, immobilized DNA on a
circular punch (approximately 1.2 mm in diameter) can be used directly as a DNA
template source.

When an assay demands the highest quality DNA, the method of choice for extraction
of DNA is the organic method. Although the protocol generally is more time-
consuming and laborious, the purity of the extracted DNA usually is higher than other
methods. More protein is removed from the DNA molecule during organic extraction
compared with other methods, and, when coupled with a filtration wash, many
enzymatic inhibitors are removed.
The majority of biological evidence analyzed in a United States forensic laboratory is
derived from rape cases. The evidence from rape cases, such as vaginal swabs and
stained clothing, most often contains nucleated cells from the male contributor (i.e.,
predominately sperm) and the female victim (i.e., epithelial cells). Elucidation of the
individual contributors’ DNA profiles can, at times, be complicated in these mixtures.
However, sperm cells can be separated from other cells during extraction. Sperm cell
membranes contain thiol-rich proteins and are resistant to cell lysis in the absence of a
chemical known as a reducing agent. The lack of use of a reducing agent results in a
differential lysis in which non-sperm cells preferentially burst. The non-sperm DNA
is released into the supernatant, creating a fraction that predominately contains DNA
from the epithelial cells from the female contributor. Subsequently, in the presence of
a reducing agent, DNA from the intact sperm can be extracted separately from the
female cells. The process purifies and enriches each fraction, so that DNA profiles
from both male and female contributors in a rape case sample can be more readily
identified.

Cetyltrimethyl ammonium bromide (CTAB)