Biology Reference
In-Depth Information
(a)
(b)
(c)
(d)
Remove
swab
Vortex and
centrifuge
Add
chloroform
Add phenol
Vortex and
centrifuge
Precipitate
At interface
Repeat 2-3 times
Add fresh phenol to a
clean tube and transfer
the aqueous phase (upper)
Figure 4.3
DNA extraction from a buccal swab cells using a salting-out method based on phenol-
chloroform. (a) Cellular material is added to a lysis buffer and proteinase K and incubated at 56
◦
C
for at least 15 minutes. (b and c) The swab is removed and phenol is added, the solution is then
vortexed and centrifuged. Precipitated protein and carbohydrate form a pellicle at the interface;
this step is repeated until there is no visible material at the interface. Protocols vary - some use
only phenol, others phenol and chloroform (isoamyl alcohol may be added to the phenol/chloroform
mixture to prevent it separating). (d) In a final step chloroform alone is added; this removes
any residual phenol, which would inhibit downstream processes such as PCR. The aqueous phase
now contains DNA. This can be concentrated by adding sodium acetate and either ethanol or
iso-propanol to precipitate the DNA, followed by centrifugation (the DNA will precipitate and form
a pellet) or by using filter centrifugation, which is similar to the steps in Figure 4.2g-f, except
that the membrane acts as a molecular sieve - allowing small molecules to pass through while
retaining DNA strands
extractions are very simple to perform and do not require multiple tube changes,
thus reducing the possibility of sample mixing [13 - 19]. The technology also pro-
vides a simple and relatively inexpensive method for long-term storage of DNA,
removing the requirement for refrigeration.
DNA extraction from challenging samples
The extraction of the many samples encountered in the forensic laboratory, including
blood and shed epithelial cells, can be carried out routinely using any of the above
techniques. There are however some sample types that necessitate variations on the
basic techniques.
Semen
Semen is one of the most commonly encountered types of biological evidence. The
extraction of DNA from the spermatozoa is complicated by the structure of the
spermatozoa (Figure 4.5). DNA is found within the head of the spermatozoa that
