Species Diagnostics Protocols: PCR and Other Nucleic Acid Methods

410 Pages·1996·26.493 MB·English

by  Justin P. Clapp

CHAPTER 1
Isolation and Purification
of Plant Nucleic Acids
Genomic and Chloroplast DNA
Mien T. G. van de Ven, Patrick G. Lanham,
and Rex M. Brennan
1. Introduction
The use of molecular protocols has expanded into virtually all branches
of plant science in recent years, and crucial to thesep rotocols is the effec-
tive isolation of plant nucleic acids in a purified state. Isolation of DNA
from plant tissue must be simple, rapid, inexpensive, reproducible, and
efficient, particularly when many samples are required, e.g., in popula-
tion studies. The increasing use of polymerase chain reaction (PCR)-
based technology in plant molecular biology, especially in transgenic
analysis, genetic mapping for genome analysis, and genetic fingerprint-
ing, has placed further emphasis on the need for the efficient isolation of
pure DNA, often from small amounts of plant tissue.
Isolation of highly purified plant DNA is often complex, particularly
from plant tissues high in polyphenolic compounds that can react with
cellular enzymes during the extraction procedure to render the DNA
unsuitable for further analysis. Contamination with polysaccharides,
mainly pectins, is also a frequent problem; the following protocols are
designed to limit their effects.
Protocols for both genomic and chloroplast DNA (cpDNA) are given.
CpDNA is now widely used, particularly in association with restriction
From Methods In Molecular B/ology, Vol 50. Species Dlagnostm Protocols PCR and Other
Nuclerc Acrd Methods Edlted by. J P Clapp Humana Press Inc , Totowa, NJ
1
2 van de Ven, Lanham, and Brennan
analysis, in phylogenetic and other studies. In most higher plants the
cpDNA is a circular molecule highly conserved within each species. As
with genomic DNA, the isolation procedures must be reliable and rapid
to give high quality cpDNA. In both cases, the starting material should
be young, soft plant tissue for the best results.
2. Materials
Horizontal gel electrophoresis equipment including gel tank, power
pack, and combs are required for all methods.
2.1. Genomic DNA
2.1.1. Method 1
1. Mortar and pestle.
2. 15-mL Centrifuge tubes.
3. Micropipets.
4. Platform shaker.
5. Benchtop centrifuge.
6. Mtcrofuge.
7. Microfuge tubes.
8. Muslin.
9. Extraction buffer: 1 or 2% CTAB (see Notes 1 and 2); 100 mM Tris-HCl,
pH 8.0,20 mA4EDTA, pH 8.0, 1.4MNaCl.
10. TE buffer:10 mMTrts-HCI, pH 8.0, 1 mMEDTA, pH 8.0.
2.1.2. Method 2
1. Blender.
2. Centrifuge (medium to low spin).
3. Microfuge.
4. Microfuge tubes.
5. Micropipets.
6. Cheesecloth.
7. Miracloth (Calbiochem, La Jolla, CA).
8. Extraction buffer: 100 miW Tris-HCl, pH 7.5, 0.35M sorbitol, 5 rnIvI
EDTA, 20 m&f sodium bisulfate added fresh just before use.
9. Nuclei lysis buffer: 200 mA4 Tris-HCl, 50 mM EDTA, 1M NaCl, 2%
CTAB, pH 7.5.
2.1.3. Method 3
1. Mtcrofuge.
2. Microfuge tubes.
Plant Nucleic Acids 3
3. Disposable grinders.
4. Vortex mixer
5. Extraction buffer: 200 rmI4 Tris-HCl, pH 8.0, 250 mM NaCl, 25 mA4
EDTA, 0.5% SDS.
2.2. Chloroplast DNA Isolation
1. Scalpel.
2. Waring blender or other blender.
3, Funnels.
4. Nylon gauze of loo-, 64-, and 30-pm pore size.
5. Glass beakers.
6, Centrifuge tubes, 50-, 15-, and 5-mL.
7. Centrifuge, medium to low spin.
8. Ultracentrifuge with SW-28 and SW-50 rotors.
9. Soft pamt brushes.
10. Pasteur pipets.
11. Micropipets.
12. Syringe and needle.
13. Dialyses tubing.
14. Flask, 1.5 L.
15. Extraction buffer: 50 mA4 Tris-HCl, pH 8.0, 7 m&I Na2EDTA, 0.35M
sucrose 5 rnA4j3-mercaptoethanol (BME) (see Note 3) (add after buffer has
been autoclaved and cooled), 0.1% bovine serum albumin (BSA) (add
after buffer has been autoclaved and cooled).
16. Wash buffer: 50 mMTris-HCl, pH 8.0,20 mMNa2EDTA, 0.35M sucrose.
17. Sucrose buffers: 50 mMTris-HCl, pH 8.0,7 mMNa,EDTA, adjusted with
sucrose to the required percentage of 20,45, or 60.
18. TE: 10 mMTris-HCl, pH 8.0, 1 mMNa2EDTA.
19. Proteinase K: 10 mg/mL, dissolved in sterile distilled water.
20. Sarcosine: lo%, dissolved in 50 mA4Tris-HCl, pH 8.0,20 mMNa,EDTA.
21. Phenol: phenol saturated with O.lM Tris-HCl, pH 7.6.
22. Phenol/chloroform/isoamyl alcohol: Mix equal amounts of chloroform/
isoamyl alcohol (24: 1) and phenol.
23. 3M Sodium acetate.
24. 100% Ethanol.
25. 70% Ethanol.
26. Cesium chloride.
27. 1% Ethidium bromide.
28. Butanol.
4 van de Ven, Lanham, and Brennan
3. Methods
3.1. Genomic DNA
One of the major considerations when isolating DNA from plant tis-
sue is the variety of plant species that exist, so that a method which works
well for one species may not work well for another. The methods
described here are generally applicable, but for each new species optimi-
zation of the DNA extraction procedure is usually required. With this in
mind, some variations on the methods are included to give greater scope
in their application. All materials and solutions should be sterilized before
use in each case.
3.1.1. Method 1
This method (1,2) uses the cationic detergent hexadecyltrimethyl-
ammonium bromide (CTAB), which is widely used for a variety of plant
tissues to disrupt cell and nuclear membranes, releasing DNA and other
components (‘3). Differences in the solubilities of nucleic acids and
polysaccharides in the presence of CTAB assists in eliminating polysac-
charide contamination. Additionally CTAB can be used to precipitate
DNA when the NaCl concentration is <0.7A4 (I).
Protecting the DNA from degradation by native nucleases or second-
ary compounds is of primary importance. For this reason, ethylene-
diaminetetraacetic acid (EDTA) is included in most DNA extraction
buffers to chelate divalent cations such as Mg2’ or Ca2+,w hich are cofac-
tors for many DNA-degrading enzymes. However, at least one DNase
that is stimulated by EDTA has been observed (4). The reducing agent,
P-mercaptoethanol, is included in extraction buffers to protect the DNA
against quinones, disulfides, peroxidases, and polyphenoloxidases (5),
whereas the inclusion of polyvinylpyrrolidone (PVP) decreasest he effect
of polyphenols, quinones, and tannins.
A contaminant, usually thought to be polysaccharide, may copurify
with the DNA. This can cause problems in dissolving the DNA or in
subsequent manipulations such as digestion with restriction endonu-
cleases. Adoption of alternative precipitation procedures to those given
in the main Methods (see Notes) may overcome this. Protein contamina-
tion is removed by chloroform extraction stages.
1. Grind material (0.5-2.0 g) in liquid nitrogen( seeN ote 4) using a mortar and
pestle. The addition of sterile sandm ay aid tissue disruption (seeN ote 5).
Plant Nucleic Acids 5
2. Add 5 mL of extraction buffer (see Note 6) and transfer to a sterile centri-
fuge tube containing 300-500 mg of insoluble PVP (see Note 7). Do not
allow the ground material to thaw out prior to the addition of extrac-
tion buffer.
3. Incubate in a water bath at 65°C for 30-60 min with occasional gentle
mixing by inversion of the tube.
4. Add an equal volume, 7.5 mL, of chloroform/isoamyl alcohol (24: 1) and
mix for 15 min by laying the tubes in a horizontal position on a platform
shaker. It is important that the phases receive thorough mixing without
severe agitation, otherwise mechanical shearing of the DNA will occur.
5. Centrifuge at 4000g for 6 min. Usually, this results in the organic and aque-
ous phases being separated by a solid plug of debris that should be left
undisturbed.
6. Filter the aqueous phase through sterile muslin.
7. Add an equal volume (approx 3 mL) of ice-cold propan-2-01 and mix by
inversion. Stand at room temperature for 15 min.
8. The precipitated DNA can either be hooked out with a sterile glass hook or
pelleted by centrimgation at 4000g for 20 mm. Occasionally, DNA is not
visible in the tube after the addition of propan-2-01; if this occurs, DNA
may still be recovered by centrifugation.
9. Wash the pellet once with 70% ethanol, to remove residual CTAB and
NaCl. Dry the pellet under vacuum and redtssolve in 200-500 pL of sterile
TE buffer. Care should be taken not to overdry the pellet, as this can lead
to problems m dissolving the DNA. Drying until no ethanol is visible usu-
ally is sufficient.
10. The DNA can be treated, if required, with RNAse (final concentration 10
pg/mL, 30 min at 65°C) followed by precipitation with 5 vol of ice-cold
100% ethanol.
3.1.2. Method 2
This method (6,7) first isolates nuclei and then lyses them to extract
the DNA. The quantities given are for relatively large amounts of leaf
material but can be scaled down (8).
1. Blend 1O -20 g of leaf tissue in a blender using 150 mL of ice-cold extrac-
tion buffer. Blending should be sufficient to homogenize the tissue (usu-
ally about 15 s), but too much blending will result in mechanical shearing
of the DNA and should be avoided.
2. Filter through two layers of sterile cheesecloth and one layer of sterile
miracloth into a precooled 250-mL centrifuge bottle on ice.
3. Centrifuge at 4°C at 750g for 5 min.
6 van de Ven, Lanham, and Brennan
4. Discard the supernatant and resuspend the pellet in 5 mL of extraction
buffer (see Note 8). Keep on ice. Add 5 mL of nuclei lysis buffer and then
2 mL of 5% sarkosyl.
5. Transfer to a 50-mL centrifuge tube.
6. Incubate for 15-20 min m a water bath at 60°C with occasronal gentle
mixing by inversion of the tube.
7. Add 15 mL of chloroform/lsoamyl alcohol (24: 1) and mtx as m step 4 m
Method 1 (see Note 9).
8. Centrifuge for 15 mm at 400g.
9. Carefully pipet the aqueous phase mto another centrifuge tube and add
2/3-l volume of propan-2-01. Mix by inversion and recover the DNA as
in steps 8-10 in Method 1 (see Notes 10 and 11).
3.1.3. Method 3
The advent of PCR technology has meant that small quantities of DNA
may be sufficient for a given experiment. The following method permits
the isolation of PCR-amplifiable DNA from small quantities of leaf
material (9). The short time required to complete this procedure makes it
possible to process many samples quickly.
1, Pinch out a section of fresh leaf material using the lid of a sterile 1.5-mL
Eppendorf tube.
2. Macerate the tissue at room temperature in the Eppendorf tube using a
disposable grinder for approx 15 s.
3 Add 400 pL of extraction buffer and vortex for 5 s. At this stage the mix-
ture can be left at room temperature for at least 1 h.
4. Centrifuge for -14,000g for 1 min and transfer 300 pL of the supematant
to a fresh tube.
5. Add 300 l.tL of cold propan-2-01, mix, and leave at room temperature
for 2 min.
6. Centrifuge for 5 min, discard the supematant.
7. Wash the pellet once with 300 PL of 70% ethanol. Centrifuge at top speed
for 5 mm in an Eppendorf centrifuge.
8. Discard the supematant, dry the pellet as m step 9, Method I, and redis-
solve it m 100 pL of TE buffer.
3.2. Chloroplast DNA Isolation
Chloroplast genomes have been studied in a wide variety of plant spe-
cies. In most cases chloroplast DNA (cpDNA) consists of a single circu-
lar molecule, which is highly conserved in size and gene arrangement.
Plant Nucleic Acids 7
The size of the plant chloroplast DNA genome ranges from 83-292 kb
(IO), whereas in land plants the variation is even smaller, 120-2 17 kb (1 I).
This size difference seems mainly owing to the size variation of a single
large inverted repeat that can be absent, as in a group of legumes and
several conifer species, or can vary in length up to 76 kb (11-13). The
genes encoded on the chloroplast genome are mostly involved in photo-
synthesis or chloroplast protein synthesis (14,15). The organization,
structure, evolution, and inheritance of chloroplast genomes have been
reviewed by Palmer and others (11,15-19). With the study of chloro-
plasts from many plant species, a variety of methods have been devel-
oped for the isolation of cpDNA (10,20).
The procedure given here has been used successfully for the isolation
of cpDNA from tobacco and potato and is a compilation of methods
described for tobacco and petunia (II), Atriplex (121, and potato (13).
This method yielded -1.5 ug cpDNA/g fresh weight of plant material,
and the cpDNA readily could be cut with restriction enzymes.
If this method is not successful, and if cpDNA isolation is impos-
sible, some suggested modifications and alternatives are given in Sec-
tion 3.2.5.
As mentioned previously, many different protocols for the isolation of
cpDNA exist. In the majority of these, intact chloroplasts are first iso-
lated, from which the cpDNA is extracted. It is also possible to extract
total cellular DNA and purify the cpDNA by CsCl-gradients (I 1). Fresh,
young, and healthy leaves, sometimes grown at low light intensity or
dark (to prevent stareh accumulation) seem to be a prerequisite for good
chloroplast yields (10,20), although cell suspension cultures (21) and
freeze-dried material (22,231 have also been used for the isolation of
cpDNA. An advantage of using freeze-dried material is that it can
improve the yield of cpDNA from, for example, wheat and rice, and it
can be stored for a long time. It also has some disadvantages, such as
requiring the use of hazardous solvents, and there seems to be an inverse
correlation between the yield of cpDNA and contamination with nuclear
DNA (23). The isolation procedures of chloroplasts from fresh leaves
basically are modifications of the methods developed by Kolodner and
Tewari (241, using DNAse I digestion of nuclear DNA and cpDNA from
broken chloroplasts, and Tewari and Wildman (25) using a sucrose gra-
dient to purify chloroplasts.
8 van de Ven, Lanham, and Brennan
Leaf tissue is briefly homogenated in extraction buffer in a blender, so
the cells are broken but the chloroplasts stay intact. The extraction buffer
contains the following components:
1. A buffer to keep the pH stable, usually Tris-HC1 but N-(2-Hydroxyethyl)-
plperazine-N’-(2-ethanesulfonic acid) (HEPES) (29) and 2-(N-morph-
olme)-ethanesulfonic acid (MES) (201 have been used.
2. An osmoticum, such as 0.3M sucrose, sorbitol (IO), or mannitol (24).
3. An oxidizmg agent, such as BME or DTT. DTT would probably be pre-
ferred to BME as it has a greater redox potenttal, IS capable of suppressing
degradative enzyme activity (26), and IS less harmful.
4. EDTA and BSA.
The following components are sometimes added to aid the chloroplast
extraction:
1. Spermine and spermidine to stabilize the plastld membrane and reduce
nuclease degradation of plastld membrane (26,27).
2. Polyethylene glycol (PEG) and or PVP have beena ddedw hen tissues are
expected to contain lots of tannins or other secondary compounds (20,28).
Chloroplasts are isolated from cell debris by filtration through several
layers of cheesecloth, miracloth, or nylon gauze up to 20-pm pore size.
To concentrate the chloroplasts they are pelleted by centrifugation
(1000-l 5OOg) and resuspended in wash buffer, usually containing the
same components as the extraction buffer, except for the BME/DTT
and BSA.
The chloroplasts can be further purified by DNAse I digestion, sucrose
gradients, or can be lysed immediately to further simplify the method
(29,30). For lysis of the chloroplasts pronase (J&27), proteinase K
(26,28,31), SDS (26,29), sarkosyl (10,2.5,27,31), Triton X-100 (23,28),
CTAB (30), or a combination of two of these lysmg agents have been
used. Some plants react differently to the various lysing agents: e.g.,
clover chloroplasts are lysed incompletely by pronase, proteinase K, and
Triton X- 100, but completely by CTAB (30).
The cDNA is either purified by phenol/chloroform extractions or CsCl
gradient. The latter tend to give purer cpDNA than phenol/chloroform
extractions, but are more laborious and time consuming. The cpDNA is
precipitated with salt and ethanol, washed with ethanol, and resuspended
in a small volume of TE buffer.
Plant Nucleic Acids 9
3.2.1. Isolation of Intact Chloroplasts
Steps l-l 1 are done in a cold room at 4°C. (If no cold room is avail-
able the work should be conducted on ice as far as possible.) All solu-
tions and materials are sterilized before use.
1. Leaves (20-30 g) are washed respectively in cold tap water and cold sterile
distilled water, drained on sterile paper tissues, and the midrib cut out with
a sterile scalpel.
2. Homogenize the leaves in a Waring blender in 100 mL of extraction buffer
by giving a few short pulses at top speed. This should disrupt the cells but
keep the chloroplasts intact.
3. Filter the homogenate through two layers of nylon gauze of IOO-pm pore
size, two layers of gauze of 64 pm, and two layers of 30 pm, respectively.
The collected filtrate should be free of most cellular debris.
4. Transfer the filtrate to 50-mL centrifuge tubes and give a slow spin of
-50g for 2 min at 4OC in a Universal table centrifuge. This should pellet
the nuclei and the chloroplasts will stay in the supernatant.
5. Pour the supernatant in a clean 50-mL centrifuge tube and spin for 10 min
at -1500g and 4°C to pellet the chloroplasts.
6. Resuspend the pellet in 10 mL of wash buffer using a soft paint brush.
7. Make a stepwise sucrose gradient by layering the followmg sucrose solu-
tions in a 50-mL centrifuge tube: 10 mL 60% sucrose buffer, 10 mL 45%
sucrose buffer, 10 mL 20% sucrose buffer. Disrupt the interfaces with a
pipet or by freezing and thawing the gradient a few times, to prevent tight
packaging of the chloroplasts.
8. Load the resuspended chloroplasts on top of the sucrose gradient and spin
for 50 min at 85,500g (Beckman ultracentrifuge with SW-28 rotor) and
4°C. A green layer of chloroplasts is formed at both interfaces, the top one
containing Intact cup-shaped chloroplasts and the second one containing
mostly round chloroplasts. The pellet contains most of the nuclear material
and starch.
9. The green bands are removed with a pipet and can either be combined or
kept separate.
10. Dilute the chloroplast solution with 3-10 vol of wash buffer and pellet the
chloroplasts by a 15-min spin at -2000g and 4°C.
11. Resuspend the pellet in 2 mL wash buffer. Option: Solution can be checked
for intact chloroplasts by examining a drop with a light microscope.
3.2.2. Isolation of Chloroplast DNA
1. Add 0.1 vol of 10 mg/mL proteinase K, mix by gently inverting the tube,
and leave at room temperature for 2 min.
10 van de Ven, Lanham, and Brennan
2. Add 0.5 mL of 10% sarcosme, mix by gently inverting the tube, and leave
at room temperature for 30 min to lyse the chloroplasts.
To purify the cpDNA either use phenol extraction (Section 3.2.3.) or
put the lysate through a cesium chloride (CsCl) gradient (Section 3.2.4.).
3.2.3. Purification of Chloroplast DNA by Phenol Extraction
1, Extract the lysate with phenol by adding an equal volume of phenol (2.5 mL),
mix by inverting the tubes several times, and spin for 10 mm at -2200g.
2. Collect the upper layer, takmg care not to include any of the interface, and
repeat the phenol extraction.
3. Extract the cpDNA solution once more with phenol/chloroform/isoamyl
alcohol.
4. Precipitate the cpDNA with 0.1 vol of 3M sodium-acetate and 2.5 vol of
100% cold ethanol.
5. If strings of DNA are formed they are hooked out; otherwise pellet the
DNA by a 10 minute spm at -3000g.
6. Wash the DNA twice with 70% ethanol and air dry.
7. Resuspend the DNA in 200 pL TE.
3.2.4. Purification of Chloroplasts DNA by C&l-Gradient
1. Add to the lysate 4.76 g of CsCl and adjust the volume to 5 mL with TE.
Dissolve the CsCl by gently inverting the tube at room temperature.
2. Add 5 pL of 1% ethidium bromide solution and spin for 12-16 h at
40,000 rpm (Beckmann ultracentrifuge with SW-50-IB rotor). A bright
pink band of cpDNA is formed at a buoyant density of = f 1.69, whereas
the pellet contains RNA.
3. Remove the cpDNA band with a syrmge and needle under ultravrolet light
(UV). If the centrifuge tubes are sealed make sure to make a hole in the top
before removing the band, to avoid creating a vacuum.
4. Extract the DNA with an equal volume of butanol saturated with water
several times, to remove all ethidium bromide from the DNA-face (bot-
tom layer).
5. Transfer the DNA solution to dialysis tubing soaked in sterile distilled
water and dialyse against 1 L of TE at 4°C. Change the TE several times
over a period of 16 h to remove the CsCl.
6. Precipitate the cpDNA as mentioned in Section 3.2.3., steps 4-7.
3.2.5. Alternative Procedures for Detecting cpDNA Variation
Chloroplast DNA variation is widely used to investigate inter- or intra-
specific relationships among plants in studies of evolution, biosystemat-
its, phylogeny, introgression, or cpDNA inheritance. Chloroplast DNA