Biology Reference
In-Depth Information
Step 1:
Sample Preparation
Decontamination
Reduction
Step 2:
DNA Extraction
Cell lysis
Extraction
(phenol:chloroform)
Extraction
(silica)
DNA extract
Step 3:
DNA Amplification
Polymerase chain reaction (PCR)
Step 4:
DNA Sequencing
DNA sequencing
FIGURE 16.4
Simplified steps for DNA extraction and sequence analysis from hard tissue.
a DNA “eliminating” product, such as household bleach. Second, the sample is reduced to
the smallest bits possible with the purpose of exposing as much surface area as possible.
This can be done by dipping the sample in liquid nitrogen to flash freeze it and then shatter-
ing the sample, particularly if the sample is soft tissue. Another option is to mechanically
reduce the sample using sandpaper, a rotary grinding tool, a hammer, or even a coffee
grinder (depending on the tissue source). A final option is to chemically reduce the sample
using a demineralization protocol.
STEP 2: DNA EXTRACTION
With the DNA sample now reduced to small particle sizes, it is ready to undergo a series of
chemical reactions designed to break open cell membranes and isolate DNA from all other
cellular components. The two most common DNA extraction methods are based on the
use of phenol and chloroform, or silica (or both). Standardized protocols for DNA extraction
exist and are widely published; an authoritative source is
Ausbel et al. (2002)
.
The process of DNA extraction produces a good amount of DNA, but that quantity is not
really sufficient for many subsequent procedures. This is the case particularly if only a trace
amount of DNAwas present in the sample in the first place, such as with ancient or forensic
DNA. Therefore it has become almost routine procedure to augment the sample through an
“amplification” process.
