A Review of Plant Ex-Amp Extraction Buffer Set
Introduction: The Plant Ex-Amp Extraction Buffer Set, along with two competitors’ one-step
DNA extraction systems, was tested for its ability to effectively produce DNA template material for
the amplification of a molecular marker in freshwater chlorophytes (green algae).
Background: Due to their great diversity and oftentimes indistinguishable morphology, algae have
traditionally been very difficult to describe and classify. It is estimated that up to a million or more
species of algae exist, only a fraction of which has currently been discovered and described. A
major area of algal study has thus focused on the identification of DNA barcodes – easily
amplifiable regions of DNA that are universally present within a certain taxonomic division and
have the right amount of variability to distinguish species – which may help with the identification
or discovery of species of algae. Extensive study has been conducted for one of the most wellknown groups of algae, the freshwater-inhabiting chlorophytes or green algae, in an attempt to find
the best barcode region. One major region – the ITS, or internal transcribed spacer portion of the
ribosomal RNA genes – has been identified as a potentially suitable molecular barcode for most
green algae, following its successful use in diatoms, another lineage of algae.
One major difficulty in this line of research, however, is the extraction of DNA from algae for
amplification and eventual sequencing. A variety of methods have been tested and used, but they all
have appreciable drawbacks. A “manual” system for DNA extraction, involving cellular lysis with a
detergent such as CTAB followed by chloroform extraction and ethanol precipitation, is very
commonly used, but it requires lots of consumables and is very time-consuming. Spin-column based
extraction methods, usually produced in kits for utilization, have been more recently utilized;
however, while throughput is increased, the procedure still remains difficult and expensive. The
most recent development in DNA template preparation has been the advent of single-step buffer
systems that quickly and easily extract DNA from cells and yield a buffer in the same volume,
without multiple pipetting, transferring, centrifuging, or eluting steps. The only major drawback of
such methods is that they normally necessitate the use of a specially-engineered robust enzyme that
can tolerate inhibitors which might be present in the unpurified buffer. Therefore, for the best
application of such buffer systems, it is essential that they are verified to produce a high-quality
template that will not inhibit the reaction of any polymerase chosen for amplification.
Methods: Two strains of freshwater chlorophytes, named JIAC3 and JIAC6, were isolated from
wild-collected water samples in the Sacramento area. Light microscopy techniques were able to
identify JIAC3 as a species of Eudorina, and JIAC6 as a species of Ankistrodesmus. Clonal cultures
were started from single cells isolated with micropipettes; JIAC3 was maintained in a liquid
medium consisting of a soil-water and fishmeal extract, and JIAC6 was maintained on agar slants
with a commercial mineral-based algal medium.
The primers used in this experiment (forward [ITS1] 5’ GGTTTCAAAGCTYTWCGTGC 3’ ;
reverse [ITS4] 5’ GTAAATACCACGGCTACGRTCTT 3’) were those first described by White et
al. (1990). Three buffer systems (including the E.Z.N.A. Plant Direct PCR Kit) were used to extract
DNA from the algae and prepare templates. The algae from each culture was harvested and
centrifuged down at 10,000 rpm x 2 minutes, and 5 µL of concentrated algal biomass was used as
starting material. Manufacturer’s instructions were followed for the preparation of each buffer, but
the volumes of each component were appropriately scaled up or down to yield a final template
volume of 45 µL (40 µL of buffer ingredients + 5 µL algal biomass).
PCRs were set up in 25 µL volumes. For amplification, FlashTaq HotStart polymerase (Empirical
Bioscience), a standard chemically-based hot-start Taq polymerase with no additional modifications
for increased robustness, was employed. Each reaction consisted of 12.5 µL FlashTaq master mix,
0.4 µM each primer, 0.5 µL template, and water to 25 µL. The PCR programs used were: 1 cycle of
94C for 3 minutes; 35 cycles of 94C for 30 seconds, annealing temperature specific to each primer
set for 20 seconds, and 72C for the appropriate duration for each primer set; followed by a final
extension cycle of 72C for 7 minutes and an infinite hold at 4C. PCR was performed with a
GeneAmp 9600 (Perkin Elmer), and products were run on a 1.2% agarose gel with TAE buffer
using the MyGel mini eletctrophoresis system and visualized with SmartGlow safe stain and the
SmartDoc 2.0 visualization system (Accuris Instruments).
Results and Discussion:
Ex-Amp Competitor 1 Competitor 2
Figure 1: Visualized gels of PCR products amplified using one of three single-step DNA extraction
buffer systems. For each pair of reactions, the left one is amplified from JIAC6, and the right one
from JIAC3. The molecular-weight marker used is the 100 bp DNA Ladder H3 (Caisson Labs).
With the listed parameters and cycling conditions, it was possible to amplify the ITS2 gene
successfully from both JIAC3 and JIAC6. Of the two, the strain JIAC3 appeared to be easier to
amplify from, both in terms of the number of successful reactions and the intensity of the product
bands for these reactions. This is possibly due to the larger, rounder cells present in each colony of
JIAC3, as opposed to the thin, small cells of JIAC6, which may have presented a greater challenge
for the buffers to extract DNA from. However, both the Ex-Amp and Competitor 2 buffer systems
were able to successfully amplify products from both JIAC3 and JIAC6. Competitor 1’s buffer,
which is comparable in terms of ease of preparation but is specifically intended for use with robustengineered enzymes, understandably did not perform as well with the polymerase and samples used
for this experiment, only amplifying a product from JIAC3.
When evaluating the other two buffers, it appears initially that they demonstrate comparable levels
of success, both having amplified the same products successfully. However, there are a few factors
which contribute to the advantage of using the Ex-Amp buffer as opposed to the competitor’s
alternative. First of all, the reaction bands from the Ex-Amp buffered PCRs are much more intense
than those of the competitor’s system. This suggests that the Ex-Amp buffer was able to extract
DNA more effectively from the same given amount of biomass. Another group of related factors
concerns the ease of the buffer setup, which the Ex-Amp system has a definite advantage in. While
the competitor’s buffer relies on a two-step heating system, implying a proteinase K digestion at
first, the Ex-Amp system only requires a single heating step of 10 minutes at 95C. This single-step
process also means that the time for preparation is cut by around two-thirds, compared to the buffer
preparation from competitor 2. The Ex-Amp system also is offered in a single reagent, as opposed
to several that must be mixed and prepared for each extraction. Not only does the Ex-Amp system
save the hassle of several liquid transfer steps, but its single buffer also makes it one of the most
scalable buffer systems – the volumes of cells used for extraction, as well as the total amount of
template produced, can easily be adjusted without having to worry about adjusting the quantities of
multiple reagents. This is especially important for running PCR for a wide diversity of samples
without too many repetitions of each, as the Ex-Amp system is also the most expensive of the three
tested, in terms of both the absolute price per kit and the relative price per volume of buffer. The
only other disadvantage of this system noted was that with the data combined from testing two other
molecular barcode regions (not shown), competitor 2’s buffer was able to amplify more different
products from the strains of algae, but the Ex-Amp universally outperformed it in terms of yield per
reaction.
In conclusion, the Plant Ex-Amp Extraction Buffer Set is an excellent product that greatly improves
upon the traditional spin-column or chloroform-ethanol type DNA extractions typically used for
plants and algae. It offers greater speed, scalability, convenience, and versatility than the older
methods, and it competes very favorably against other products intended for the same purpose.
Although its greatest disadvantage is its price, the Ex-Amp system is highly efficient at extracting
DNA, which still makes it potentially very useful for preparing template material from low quantity
or quality samples, doing large scale screenings over a wide diversity of sample types or species,
and preparing small quantities of template with the highest reproducibility.