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Abstract
Taxonomists must be familiar with a number of issues in collecting and transporting samples using freezing
methods (liquid nitrogen and dry ice), desiccants (silica gel and blotter paper), and preservatives (CTAB,
ethanol, and isopropanol), with each method having its own merits and limitations. For most molecular
studies, a reasonably good quality and quantity of DNA is required, which can only be obtained using
standard DNA extraction protocols. There are many DNA extraction protocols that vary from simple and
quick ones that yield low-quality DNA but good enough for routine analyses to the laborious and time-
consuming standard methods that usually produce high quality and quantities of DNA. The protocol to
be chosen will depend on the quality and quantity of DNA needed, the nature of samples, and the presence
of natural substances that may interfere with the extraction and subsequent analysis. The protocol described
in this chapter has been tested for extracting DNA from eight species and provided very good quality and
quantity of DNA for different applications, including those genotyping methods that use restriction
enzymes.
Key words Blotter paper, CTAB, Desiccant, DNA extraction, Dry ice, Ethanol, Leaf sampling,
Silica gel
1 Introduction
Pascale Besse (ed.), Molecular Plant Taxonomy: Methods and Protocols, Methods in Molecular Biology, vol. 1115,
DOI 10.1007/978-1-62703-767-9_3, © Springer Science+Business Media New York 2014
53
54 Kassa Semagn
and (c) FTA card (Whatman Inc., Clifton, NJ). FTA card is a paper
specially treated with chemicals that inactivates pathogens, pro-
tects the DNA from degradation, and allows the cards to be stored
at room temperature for extended periods of time [5]. Plant tissue
is physically crushed on the card; small discs are punched from the
card, washed with aqueous buffers, and dried; and a small disk
(1.2 or 2 mm in diameter) is used as templates for PCR (http://
www.whatman.com/References/WGI_1397_PlantPoster_V6.
pdf). For most applications, however, a reasonably good quality
and quantity of DNA is required, which can only be obtained
using standard DNA extraction protocols. Therefore, a fast, sim-
ple, high-throughput, and reliable extraction protocol for genomic
(nuclear, mitochondrial, and chloroplast DNA) or organelle DNA
from leaves, seeds [6–8], roots [9], flower petals [10], and pollen
grains [11] is a prerequisite for most molecular marker studies.
This review and protocol focuses only on leaves as most molecular
studies use leaf DNA.
Researchers must be familiar with a number of issues prior to
collecting leaf samples including the molecular technique chosen
for genotyping, the nature of the tissue to be collected (soft and
thin leaves for mesophytes, hard and/or thick leaves for xero-
phytes, and succulent leaves), the quantity of tissue to be collected,
the geographical distance of the sampling sites, anticipated dura-
tion of the collection mission, and the mode of transport from the
sampling site to the laboratory. All of these factors should be con-
sidered in order to prepare sufficient quantity of the required
materials prior to the sample collection mission.
DNA can be extracted from fresh, frozen, lyophilized, pre-
served, or dried leaf samples. Fresh or fresh-frozen leaves in liquid
nitrogen (http://en.wikipedia.org/wiki/Liquid_nitrogen) are
ideal for DNA extractions. However, it is difficult to immediately
place samples in liquid nitrogen in the field because (a) it requires
a liquid nitrogen container with a wide mouth and a leakage-proof
lid; (b) liquid nitrogen evaporates from such relatively large tank in
a shorter period of time; and (c) there are safety concerns sur-
rounding transporting liquid nitrogen in vehicles and in air planes.
Dry ice (http://en.wikipedia.org/wiki/Dry_ice) is a solid form of
carbon dioxide, and it can be used as an alternative to liquid nitro-
gen. Dry ice can be easily transported in Styrofoam cooler boxes
(http://www.mrboxonline.com) that are well sealed in packing
tape. It can last up to a week in these cooler boxes, although the
duration depends on the climatic conditions of the sampling sites,
the quantity of samples, and the thickness/insulation of the
Styrofoam boxes. There are little or no safety concerns for road
transportation of dry ice when compared to liquid nitrogen.
However, airlines are reluctant to transport dry ice. Gel pack
(http://www.coldchaintech.com/products/gel-packs.php) can
also be used for sampling and transporting plant materials from the
Tissue Sampling and DNA Extraction 55
field. They last for up to a week but their heavy weight can cause
samples to break up into pieces. Also, the cooling capacity of gel
packs is not as good as both liquid nitrogen and dry ice.
Many taxonomists rely on silica gel (http://en.wikipedia.org/
wiki/Silica_gel) to dry their plant samples [12]. This method is
generally considered effective for most species as the rate of desicca-
tion is rapid enough to prevent DNA degradation. However, the
improper use of silica gel (using too little silica gel relative to the
amount of leaf tissue and not breaking up large leaves into smaller
pieces) often results in DNA degradation that may not be suitable
for applications that requires high molecular weight DNA, such as
restriction fragment length polymorphism [13] and genotyping by
sequencing [14]. Some researchers reported the use of sodium
chloride/cetyltrimethylammonium bromide (NaCl/CTAB) pres-
ervation solution [15, 16], while others used a wide range of pre-
servative chemicals, such as ethanol, acetone, 2-propanol
(isopropanol), diethyl ether, and ethyl acetate [17, 18]. Another
option is to dry the leaves at ambient temperature by pressing them
on blotter (absorptive tissue) paper that removes the moisture from
the leaves. The blotter paper must be changed a couple of times
until the samples are properly dried. We used dry ice, ethanol, iso-
propanol, NaCl/CTAB solution, silica gel, and blotter papers for
collecting leaf samples from fields for eight species and extracted
DNA after storage up to a month at room temperature except those
samples transported in dry ice that were stored in freezers (−20 or
−80 °C) after arrival in to the lab. All the six leaf sampling methods
provided reasonably good DNA quality that work well for microsat-
ellite or simple sequence repeat (SSR) markers. However, the DNA
isolated from samples collected in dry ice, silica gel, and blotter
paper were consistently high quality (Fig. 1) than the preservative
methods for applications that require restriction digestion of the
isolated DNA. The quality of DNA isolated from NaCl/CTAB pre-
served samples was very good on agarose gels, but the ratio of
absorbance at the 260 and 230 nm in spectrophotometers was con-
sistently lower than 1.7 for all species that we tested. Such lower
ratios are indictions for the presence of contaminants (impurities)
that may affect the use of DNA for some applications.
Once the plant samples are brought into the lab, the next step
is to prepare them for DNA extraction. Depending on the pur-
pose of the research, DNA can be extracted from a single plant or
bulks of several plants. The heterogeneous nature of outcrossing
species contributes to high levels of within accession or variety
genetic variability that requires for the analysis of a large number
of representative individuals per accession. As genotyping of a
large number of individual samples per accession is costly and time
consuming, several studies have used the bulked method [19–24]
for quick molecular studies. Plant bulking or DNA pooling can be
used for such purposes. In plant bulking, researchers harvest leaf
56 Kassa Semagn
Fig. 1 The quality of DNA extracted from leaf samples collected using different options: dry ice, 100 % ethanol,
100 % isopropanol, silica gel, tissue paper, and NaCl/CTAB. Each sample (A, cassava; B, sweet potato; C,
orange; D, bougainvillea; E, banana; F, grasses; G, maize; and H, papaya) was extracted in duplicate
2 Materials
2.2 DNA Extraction 1. 1.2 mL strip tubes with strip caps or 96 deep well plates with
self-closing mats.
2. 4 mm metal grinding balls or 3 mm tungsten carbide beads.
3. Metal grinding ball dispenser (optional).
4. Plate centrifuge.
5. Shaking water bath.
6. Fume hood.
7. Dissecting scissors.
8. Dissecting forceps.
9. Lyophilizer or freeze dryer (optional).
10. Agarose gel electrophoresis facility (including gel casting trays,
combs, electrophoresis tank, and power supply).
11. Tissue grinder (e.g., GenoGrinder or Tissue Lyser).
12. DNA extraction stock solutions: 1 M Tris, pH 7.5; 0.5 M
EDTA pH 8.0 (see Note 1); 5 M NaCl; mercaptoethanol.
Mercaptoethanol must be used under fume hood.
13. CTAB.
14. CTAB DNA extraction buffer that contains 0.20 M Tris,
pH 7.5; 0.05 M EDTA, pH 8.0; 2 M NaCl; 2 % CTAB; and
1 % β-mercaptoethanol.
15. Liquid nitrogen or dry ice.
16. Single and 8-channel pipettes of different volume.
17. Pipette tips of different sizes.
18. Glass wares: beakers, flasks, reagent bottles of different sizes,
measuring cylinders of different sizes.
60 Kassa Semagn
19. Balances.
20. Weighing boats.
21. Heaters and stirrers.
22. RNase A.
23. Proteinase K (optional).
24. Ethanol (70 % and absolute).
25. Isopropanol.
26. Chloroform (must be used in fume hood).
27. Isoamyl alcohol.
28. Gloves.
29. Kleenex tissue paper.
30. Double-distilled water.
31. 0.1× TE (10 mM Tris, pH 7.5–8.3 and 0.1 mM EDTA,
pH 8.0).
32. Agarose.
33. Ethidium bromide or GelRed.
34. Electrophoresis buffers: 10× Tris–borate EDTA (TBE) or 50×
Tris–acetate EDTA (TAE).
35. Size markers with known concentrations—lambda (λ) DNA,
1 kb DNA ladder, and λ DNA-HindIII digest (optional).
36. Gel documentation system (gel camera, UV tray with white
and UV light).
37. Spectrophotometer or plate reader (optional).
3 Methods
3.1 Leaf Sampling Sample very young leaves from the shoot tips of a single healthy
plant or bulks of about equal leaf tissue from several healthy plants
per accession using one of the options described below. The three
leaves closest to the shoot tip are the best; the younger the leaves,
the better the DNA quality and quantities.
1. Sampling using preservatives: harvest very young leaves from
the shoot tips, fold them, and put them into a 14–15 mL test
tube. Write the sample number on a small piece of paper with
a pencil and insert this into the tube. Add about 8 mL of 100 %
ethanol, isopropanol, or NaCl/CTAB solution; close the caps
tightly; and store at room temperature.
2. Sampling using silica gel: harvest very young leaves from the
shoot tips, tear or cut the leaf material into smaller pieces, and
put them into 3 × 4 inches Ziploc bags (see Note 2). Write the
sample number on a small piece of paper and insert this into the
Tissue Sampling and DNA Extraction 61
Ziploc bag. Add sufficient silica gel (deep blue in color) and
close the Ziploc bag. The ratio of leaf tissue to silica gel should be
at least 1:5, preferably 1:10. Check the Ziploc bags at a regular
interval, and replace the silica gel when its color changes from
blue to pink. Keep replacing the silica gel until the blue color
remains stable. Most leaf samples completely dry within 24 h.
3. Sampling using blotter (tissue) papers: harvest small young
leaves from the shoot tips, flatten the leaf surface without over-
lapping the leaves, and press them on to a pile of blotter papers.
Write the sample number on a small piece of paper and insert
between the blotter papers. To secure the samples, fold all four
sides of the blotter papers and staple them or insert them in
paper bags.
4. Sampling using dry ice: harvest young leaves from the shoot
tips and put them in perforated Ziploc bags (see Note 3). Write
the sample number on a small piece of paper with a pencil and
insert this into the Ziploc bags. Immediately put the Ziploc
bags with the leaf samples into a Styrofoam box containing dry
ice. To minimize evaporation of the dry ice, always keep the lid
(cover) of the Styrofoam box very tight by securing with a
packing tape.
3.2 DNA Extraction 1. Assemble strip tubes (eight tubes per strip) into racks (hereafter
referred as collection tubes) and label each strip.
2. Add two stainless steel grinding balls per tube and precool the
collection tubes in liquid nitrogen or dry ice.
3. Harvest about 100–150 mg of fresh leaf sample (from a single
plant or from bulks of several plants per accession), cut into
pieces with a scissor, and transfer (using forceps) into the col-
lection tubes (see Note 4). At all times, keep the collection
tubes and samples in liquid nitrogen or dry ice. If a freeze
dryer (lyophilizer) is available, freeze-dry the samples for 3–4
days as described in the user’s manual. For leaf samples col-
lected using one of the four options described above (preserva-
tives, silica gel, blotter paper or dry ice), use an equivalent
amount of tissue.
4. Prepare a fresh CTAB DNA extraction buffer (see Note 5) and
keep this extraction buffer in 65 °C water bath until the sam-
ples are ready for grinding.
5. Sandwich each plate containing collection tubes between
adapter plates and balance them using an ordinary weighing
balance. Fix the sandwiched plates into the tissue grinder and
grind the samples into fine powder as described in the user’s
manual. We often grind samples by shaking for 2 min at full
(1×) speed of strokes/min using a GenoGrinder-2000 or at
25 Hz with a Tissue Lyser. Remove the plates, exchange the
62 Kassa Semagn
3.3 DNA Quality 1. Assemble the gel casting trays and combs as described in the
and Quantity Checks user’s manual.
2. Prepare 0.8 % agarose gel as follows: add 0.8 g of agarose in a
250 mL flask containing 100 mL of 1× TBE or TAE buffer and
boil in a microwave oven until the agarose completely dis-
solves. Cool to about 60 °C, add 5 μL of gel stain (ethidium
bromide or GelRed) to get a final concentration of 0.5 μg/mL
of the gel volume, and mix very gently. Carefully pour the gel
mix into the gel casting tray and quickly remove air bubbles, if
any, using pipette tips. Leave the gel to polymerize for
30–60 min at room temperature (see Note 9).
64 Kassa Semagn
4 Notes
Acknowledgements
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