It used to be standard practice to shave the area around the
incision before surgery, as it was thought that hair harbored bacteria
that would cause wound infection. Beginning in the 1970s, doctors found
this was unnecessary, and in fact was associated with higher incidence
of post-operative infection. This history comes to mind in reading March
2010 BioTechniques report by researchers from University of Guelph
demonstrating that DNA suitable for PCR and sequencing can be obtained
simply by leaving specimens in alcohol overnight!
Of the three steps required to get from a specimen to a DNA
barcode, namely DNA isolation, PCR (polymerase chain reaction), and
sequencing, the first step is the most labor intensive and hardest to
automate. Numerous protocols/kits have been developed to optimize DNA
isolation from various types of specimens, such as plant vs animal
tissues. As described by the Guelph researchers, “these procedures force
cells to release their DNA via physical pertubation and/or chemical
treatment, which is then followed by a clean-up procedure in which
unwanted cellular compoents are separated from the DNA.” The researchers
“hypothesized that a small amount of DNA leaks from the tissue into the
preservation solution (usually ethanol), and that this DNA was
amplifiable using a standard PCR protocol.” To start, they analyzed
Monte Alban mescal, which is sold with a “worm” (a caterpillar of the
agave moth, Hypopta agavis) in each bottle. They evaporated 50
mL mescal, re-dissolved the residue in water, applied this to a Qiagen
MinElute spin column, resuspended the product in 50 μL water, and used 2
μL of resulting solution in a standard 25 μL PCR reaction, with
successful amplification and sequencing of 130 base mini-barcode of COI.
This case was presumably challenging as mescal is only 40% ethanol and
contains a variety of material that might inhibit PCR. In subsequent
tests, 1 mL of 95% ethanol used to preserve specimens was evaporated,
resuspended in 30 μL of water without column
purification, and 2 μL used for PCR.
By evaporating 1 mL of ethanol in which specimens had been stored
overnight (out of 2 mL total ethanol volume) and re-suspending residue
in water, Shokralla and colleagues amplified and sequenced 130-base and
650-base fragments of COI and 1100-base fragment of 28s RNA from 25
whole insect specimens (mayflies, caddisflies; 1 gave COI only) and rbcL
from 45 plant specimens (0.5 mm leaf samples). They also obtained COI
sequences by sampling 1 mL of ethanol solution from 7 insect specimens
stored at room temperature for 7 to 10 years. The researchers note this
approach could facilitate for “high-throughput” analyses, as it involves
liquid handling which is easy to automate, avoids destructive sampling,
and could be used even when “there is simply no sample left for further
analysis.” They conclude with a caution about “field sampling
procedures that include placing mixtures of specimens in an ethanol jar”
as this “may increase the chance of cross-contamination.”
The remarkably simple procedure reported by Shokralla and colleagues
offers benefits to many persons who want to get DNA barcode
identifications. I look forward to applications of this method in
research and commercial laboratories, classrooms, and perhaps kitchens!
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