Plant Molecular Biology Reporter 20: 161-175, June 2002

9 2002 International Society for Plant Molecular Biology. Printed in Canada.

Protocols

Comparison of Seven DNA Extraction and

Amplification Protocols in Historical Herbarium
Specimens of Juncaceae

L E N K A D R A B K O V A 1'*, JAN K I R S C H N E R 1 and CESTMIR V L ~ K 2

llnstitute of Botany, Academy of Sciences, Zdmek 1, 252 43 Prfthonice, Czech Republic;
2Centre for Integrated Genomics and Institute of Molecular Genetics, Academy of
Sciences, Flemingovo ndm. 2, 166 37 Prague 6, Czech Republic

Abstract. Seven DNA extraction protocols were used to obtain DNA from herbarium
specimens of Juncus and Luzula (Juncaceae) of various ages. DNA of historical samples is
difficult to extract, and the extracts are seldom of good quality. The quality of DNA ob-
tained was estimated by using a spectrophotometer to measure the A260/280 absorbance
ratio. The total DNA yield was measured by a fluorometer. The results indicate the success
of using both mixer mill grinding and a DNeasy Plant Kit. Another extraction protocol
(grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples.
Modified CTAB extraction, with a lengthy precipitation, usually provided good amounts of
DNA. Other protocols did not give satisfactory results.

Key words: cpDNA, DNA extraction, herbarium specimens, Juncaceae, Juncus, Luzula,
PCR amplification

Abbreviations: CTAB, hexadecyltrimetylammonium bromide; EtOH, ethanol.

Introduction

Herbarium collections are a potentially important source of material for phylo-

genetic studies. With the expansion of molecular techniques, the historical collec-
tions have become relatively widely used. The specimens are useful especially for
very rare or endemic plants because sometimes more plants are in the herbarium
than in nature. Most future molecular taxonomic studies will probably be partly or
entirely based on DNA extracts from herbarium specimens because of the easy
accessibility and richness of herbarium collections. This article should serve as a
tool for projects involving DNA extraction from herbarium specimens of
graminoid plants.
The problem of DNA extraction is crucial for further analyses of herbarium
samples. The satisfactory quality of DNA is essential for the success of the whole
molecular study. However, DNA isolation from dried specimens usually requires

*Author for correspondence, e-mail: drabkova@ibot.cas.cz; fax: +420 2 67050031;

ph: +420 2 71015 258.
162 Drdbkovgt et al.

some modifications to frequently used protocols (Rogers, 1994) because of the

small amount of dry herbarium tissue available.
The herbarium material is dried and stored on herbarium sheets in packages.
If the specimens are air-dried at up to 42~ (see Taylor and Swann, 1994), they
contain a useful amount of high molecular weight DNA. Air-drying is considered
to be better than the preservation of tissues in silica gel or anhydrous CaSO 4 (Tay-
lor and Swann, 1994). In general, old air-dried material that has not been treated
with chemical preservatives, high temperatures, or microwaves has the best
chance of yielding useful DNA (Taylor and Swann, 1994).
The main goal of this study is to evaluate various methods of DNA isolation
in terms of DNA yield and amplification quality. To preserve DNA well, it is
necessary to dry plants as fast as possible. Extraction results depend on how the
plant material is prepared, how many times the collection is disinfected, and the
type of chemicals or procedures used. For instance, DNA was seriously degraded
in leaves that were microwaved (Hall, 1981; Hill, 1983; Bacci et al., 1983), boiled
in water, or immersed in chemical solutions. Another important factor is the regu-
lar herbarium treatment used to keep specimens free of pests. Fumigation meth-
ods have been changed from time to time (Metsger and Byers, 1999), making it
difficult to be sure about the DNA quality. The extent of DNA degradation in her-
barium specimens appears to be related to the condition of the fresh leaf rather
than the year in which it was dried (Rogers and Bendich, 1985).
Obtaining high-quality DNA depends on the extraction technique used. Sev-
eral DNA isolation techniques that are useful for dry plant tissue from herbarium
specimens have been described (Wittzell, 1999; Ristaino et al., 2001; Rogers,
1994; Taylor and Swann, 1994). The most convenient organ to sample is the leaf.
However, in the Juncaceae, the morphological structure of the leaves (especially
monofacial leaves with sclerenchyma strands and parenchyma of rounded cells in
the centre of the leaf) is not suitable for rapid protocols, especially in Juncus.
Moreover, one of the most widespread and diverse sections, Juncus sect.
Juncotypus, has leaves reduced to brownish scales at the plant base, so DNA is
obtained from the stems.
This study is the first part of the Juncaceae phylogeny project (Phylogenetic
analysis of 2 cosmopolitan plant genera, Luzula and Juncus), for which it was
necessary to develop satisfactory methods for DNA preparation for sequencing
and other molecular techniques. Seven extraction techniques were compared using
fresh material and dry herbarium specimens collected from 1927-1998. This arti-
cle summarizes the results of a comparison of the techniques in regards to good
amplification and purity of obtained DNA.

Materials and Methods

DNA extraction

Total genomic DNA was extracted from dried leaf tissue (0.1 g usually, 0,5 g in a
few cases) from the herbarium specimens of various ages by means of the follow-
ing 7 procedures:
Juncaceae DNA extraction and amplification 163

1. DNeasy Plant Mini Kit (QIAgen)

Solutions:
9 Buffers DNeasy Plant Mini Kit (AP1, AP2, AP3, AE)
9 EtOH 100%
Equipment:
9 Mixer mill
9 Centrifuge
9 Water bath (65~
9 1.5-HL microcentrifuge tubes
Protocol:
Mechanical disruption of plant material proved to be a limiting step when han-
dling multiple samples in parallel (Csaikl et al., 1998). Therefore, the tissue was
ground in the mixer mill with ceramic cylinders (G-BIOgene). This procedure is
optimal for sufficient homogenization of hard leaf structure of Juncus species.
Extraction was performed according the QIAgen protocol with a small
modification for dried samples. For lysis of the cells, we used a longer incubation
time (30 min), 450 ~tL of AP1 buffer (instead of 400 ~tL), 50 iLL of AE buffer (in-
stead of 100 ~tL) for increasing the final DNA concentration, and a longer elution
time (10 rain). These modifications were introduced mainly in the laboratory of In-
stitute of Botany Copenhagen University (G. Petersen, personal communication).

2. Sorbitol DNA extraction with liquid nitrogen

This extraction protocol is according to Storchov~i et al. (2000), with a long pre-
cipitation in cooled isopropanol.
Solutions:
9 Liquid nitrogen
9 Extraction buffer (0.1 M Tris-HC1 [pH 7.5], 0.005 M EDTA [pH 8], 0.35 M
sorbitol, 10 nM 2-mercaptoethanol)
9 Lysis buffer (0.2 M Tris-HC1 [pH 7.5], 0.05 M EDTA [pH 8], 2 M NaC1, 2%
CTAB)
9 Chloroform-isoamylalcohol (24:1)
9 Isopropanol
9 80% EtOH
9 TE
Equipment:
9 Small mortar and pestle
9 Centrifuge
9 Thermoblock (65~
9 1.5-p.L microcentrifuge tubes
Protocol:
9 Grind the plant material (a piece - 1 0 mm X 5 mm) with mortar and pestle, add
1.3 mL of extraction buffer, transfer to 1.5-mL Eppendorf tube, and swirl.
9 Incubate at room temperature for 20 min.
164 Drdbkovci et al.

9 Centrifuge for 5 min at 7000 rpm (rcf 4500) at 20~ and split the supernatant.
9 Dissolve pellet in 0.3 mL of extraction buffer and 0.3 mL of lysis buffer, and
swirl.
9 Incubate at 15 min at 65~ in thermoblock.
9 Add 0.6 mL of chloroform-isoamylalcohol (24:1) mixture and swirl.
9 Centrifuge for 10 min at 9000 rpm (rcf 7500) at 20~
9 Transfer the supernatant (-600 mL) into a new Eppendorf tube.
9 Add 400 mL of isopropanol, mix well, and precipitate DNA for 1 h at -20~
(better results are with long-term precipitation of >3 d).
9 Centrifuge for 15 min at 13,000 rpm (rcf 15,700) at 4~
9 Add 0.7 mL of 80% EtOH and leave it overnight at -20~
9 Centrifuge for 10 min at 10,000 rpm (rcf 9300) at 4~
9 Take away the supernatant and dissolve pellet in 40 ~tL 10X TE.

3. CTAB extraction

Total DNA from leaf tissue of herbarium specimens was extracted in CTAB isola-
tion buffer according to the protocol of Doyle and Doyle (1987, 1990) as modi-
fied by Wittzell (1999). One-year-old herbarium specimens were used.
Solutions:
9 CTAB isolation buffer
9 Chloroform-isoamylalcohol (24:1)
9 Isopropanol
9 EtOH
9 TE
Equipment:
9 Small mortar and pestle
9 Centrifuge
9 Thermoblock (65~
9 1.5-~tL microcentrifuge tubes
Protocol:
9 Preheat CTAB solution to 60~
9 Grind plant material in 1.5-mL Eppendorf tube with 200 ~tL of CTAB.
9 Add 500 ~tL of CTAB and incubate at 60~ for 45 min. Mix twice.
9 Add 600 ~tL of chloroform-isoamylalcohol and mix.
9 Centrifuge for 15 min at 13,000 rpm (rcf 15,700) at 4~
9 Transfer 600 ~tL of supernatant to new Eppendorf tube, add 500 ~tL of chloro-
form-isoamylalcohol, and mix.
9 Centrifuge for 15 min at 13,000 rpm (rcf 15,700) at 4~
9 Remove 500 ~tL of the supernatant, add 500 I.tL of isopropanol and leave it at
-20~ for at least 3 d.
9 Centrifuge for 15 min at 13,000 rpm (rcf 15,700) at 4~ and leave at -20~ for
at least 1 h.
9 Centrifuge for 15 min at 13,000 rpm (rcf 15,700) at 4~ and remove the
supernatant.
9 Wash pellet, add 80% EtOH, and leave it overnight at -20~
Juncaceae DNA extraction and amplification 165

9 Centrifuge for 10 min at 10,000 rpm (rcf 9300) at 4~

9 Remove the supernatant and dissolve pellet in 40 ~tL of 10X TE.

4. Genome DNA purification GenElute T M Plant Genomic DNA Purification Kit

Solutions:
9 Liquid nitrogen
9 Lysis solution (part A + part B)
9 Precipitation solution
9 Binding solution
9 Wash solution
9 EtOH 100%
Equipment:
9 Mixer mill
9 Centrifuge
9 Water bath (65~
9 Filtration columns (Sigma)
9 1.5-~tL microcentrifuge tubes
Protocol:
Approximately 0.1 g of plant leaf was ground in liquid nitrogen. Other steps were
according to the Sigma protocol. The concentration of DNA in the second elution
extract was very low; thus, repeated elution was not applied.

5. Modified total DNA extraction with phenol purification and liquid nitrogen
Phenol was used for purification. After a 15-min incubation in the thermoblock, a
mixture of phenol and chloroform ( 1 : 1 , 3 0 0 ~tL) was added and mixed rigorously.
Other steps were the same as in the sorbitol extraction (protocol 2).
Protocols 6 and 7 are commonly used in the Jodrell Laboratory, Royal Bo-
tanical Gardens Kew. In this experiment, they were applied to a very small
amount of plant tissue (<0.1 g) for which this technique is not quite suitable. It is
usually used for herbarium samples of at least 0.2 g.

6. QIAquick extraction (to QIAquick PCR Purification Kit Protocol)

Solutions:
9 CTAB + 2 % PVP preheated solution
9 2-mercaptoethanol
9 24:1 chloroform-isoamylalcohol
9 QIAquick buffers (PB, PE, EB buffer)
9 Isopropanol
9 EtOH (70%)
Equipment:
9 Mortal and pestle
9 Centrifuge
9 Water bath (65~
9 Vacuum manifold
166 Dr~bkovd et al.

9 QIAquick spin columns

9 50-mL collection tubes
9 1.5-~L microcentrifuge tubes
9 Pasteur pipette

Protocol:
This protocol was applied according to Doyle and Doyle (1987) and modified
with the QIAquick PCR Purification Kit Protocol for extraction of total DNA.
9 Preheat 10 mL of isolation buffer (CTAB + 2% PVP) containing 80 ~tL of
2-mercaptoethanol in a 65~ water bath.
9 Grind leaf tissue with mortar and pestle (preheated to 65~ using an isolation
buffer.
9 Incubate at 60-65~ for 20 min. Mix it twice during incubation.
9 Add 500 mL of 24:1 chloroform-isoamylalcohol and incubate for 60 min. Mix
during incubation.
9 Centrifuge at 9000 rpm (rcf 7500) at 25~ for 10 min.
9 Remove aqueous phase with a Pasteur pipette and transfer to 50-mL tube.
9 Add 750-~tL of PB buffer and mix.
9 Place a QIAguick spin column in a provided 2-mL collection tube.
9 Use vacuum manifold and discard flow-through.
9 Add 0.75 mL of PE buffer and wash.
9 Use vacuum manifold and discard flow-through.
9 Place QIAquick column in a new 1.5-mL microcentrifuge tube.
9 Add 30-HL EB buffer for elution to the center of the QIAquick membrane.
9 Centrifuge at 9000 rpm (rcf 7500) at 25~ for 1 min.
9 Add two-thirds volume of -20~ isopropanol and mix gently to precipitate
DNA.
9 Store at -20~ for at least 3 d.
9 Transfer 300 ~L to new 1.5-p~L microcentrifuge tube.
9 Centrifuge at 3000 rpm (rcf 800) for 5 min.
9 Add 0.7 mL of EtOH and leave it at -20~ overnight.
9 Centrifuge for 10 min at 10,000 rpm (rcf 9300) at 4~
9 Remove the supernatant and dissolve pellet in 40 ~tL 10X TE.

7. QIAquick extraction (to QIAquick PCR Purification Kit Protocol) with long
term precipitation in isopropanol and CsCl2 gradient
Protocol:
The first stage of extraction was the same as in protocol 6. It was completed using
long-term precipitation and gradient ultracentrifugation.
DNA was extracted from 0.1 g of dried samples using the modified CTAB
method of Doyle and Doyle (1987) with QIAquick columns and precipitated with
isopropanol at -20~ for at least 2-4 wk (according to the standard Qiagen proto-
col: QIAquick PCR Purification Kit Protocol). DNA was purified by a cesium
chloride gradient.
Juncaceae DNA extraction and amplification 167

8. CTAB extraction from the fresh material

For comparison of the yield of DNA from different extraction methods we used
the same extraction as in protocol 1 for fresh material (Storchov~i et al., 2000).
The total DNA was isolated from approximately 0.7-1 g of fresh leaves (con-
served in CTAB) of the same collection of samples.
Solutions:
9 Extraction buffer (0.1 M Tris- HC1 [pH 7.5], 0.005 M EDTA [pH 8], 0.35 M
sorbitol, 10 nM 2-mercaptoethanol)
9 Lysis buffer (0.2 M Tris-HC1 [pH 7.5], 0.05 M EDTA [pH 8], 2 M NaC1, 2%
CTAB)
9 Chloroform-isoamylalcohol (24:1)
9 Isopropanol
9 80% EtOH
9 TE
Equipment:
9 Small mortar and pestle
9 Centrifuge
9 Thermoblock (65~
9 1.5-~tL microcentrifuge tubes
Protocol:
9 Grind the plant material (-10 mm X 5 mm) with mortar and pestle in 300 mL
of extraction buffer. Add an additional 300 mL of extraction buffer, mix, trans-
fer to 1.5-mL Eppendorf tube, and swirl.
9 Incubate at room temperature for 20 min.
9 Centrifuge for 5 min at 7000 rpm (rcf 4500) at 20~ and split supernatant.
9 Dissolve pellet in 0.3 mL of extraction buffer and 0.3 mL of lysis buffer and
swirl.
9 Incubate 15 min at 65~ in thermoblock.
9 Add 0.6 mL of chloroform-isoamylalcofiol (24:1) mixture and swirl.
9 Centrifuge for 10 min at 9000 rpm (rcf 7500) at 20~
9 Transfer the supernatant (-600 mL) to new Eppendorf tube.
9 Add 400 mL of isopropanol and precipitate DNA at -20~ for at least 1 h. Mix
well.
9 Centrifuge for 15 min at 13,000 rpm (rcf 15,700) at 4~
9 Add 0.7 mL of 80% EtOH and leave it overnight at -20~
9 Centrifuge for 10 min at 10,000 rpm (rcf 9300) at 4~
9 Remove the supernatant and dissolve pellet in 40 ~tL of 10X TE.

Specific PCR amplification

The first reactions (using primers for rbcL, Table 1) contained 20 ~tL of dNTP,
5 ~tL of buffer and MgC12, 14 ~tL ddH20, and 0.2 ~L Taq DNA polymerase, and
5 HL of primers (Table 1). In the second PCR reaction (trnL-trnF), 1 HL of DNA,
3.75 ~tL of dNTP, 2.5 ~L of buffer, 3 ~tL of MgC12, 12.45 ~tL ddH20, 0.3 ~tL Taq
DNA polymerase, and 0.5 ~tL of universal primers were used (Taberlet et al.,
1991) (Table 1). Conditions were optimized according to Cobb and Clarkson
168 Dr6bkovd et al.

Table 1. Primers used to amplify cpDNA.

Name of Length
primer Sequence Combination used (bp)

RH1S ATG TCA CCA CAA ACA GAA ACT RHIS/Hv362R 362
Hv362 R TGA ACC CAA ATA CGT TAC CCA
Hv234 CGT TAC AAA GGA CGA TGC Hv234/J556R 322
J556R ACA TTC ATA AAC TGC TCT ACC
Hv522 TAA ACC AAA ATT GGG ATT ATC Hv522/CYMn803 281
CGC
Z674RS GAT TTC GCC TGT TTC GGC TTG
Ce622 TCA CAA CCA T'I~TATG CGT TG Ce622/CYMn 803(R), 181,
Ce622/J949R 327
CYMn803(R) AAA CCA CCA GTT AGG TAG TC

J949R CTC CAC CAG ACA TAC GTA ATG C

Hv890 TGC ATG CAG TTA TTG ATA GAC HV890/Hv1204RS, 314,
Hv890/J! 352R, 462,
Hv890/ZI1516R 626
Z1204RS CCC TAA GGG TGT CCT AAA GTT
J1352R GCA GCA GCT AGT TCA GCA CTC C
ZI1516R AGA TGA GCC GAA TTG AAT TGC
trnL (e) GGT TCA AGT CCC TCT ATCCC trnL-trnF 380-400
trnF (f) ATT TGA ACT GGT GAC ACG AG

(1994). The Juncaceae samples were amplified as small fragments of cpDNA

(281-462 bp). A size of approximately 300 bp is best for successful amplification.
Good results depend on use of high-quality Taq polymerase. (We used Taq D N A
polymerase from Amersham Pharmacia Biotech and AmpliTaq Gold from Perkin
Elmer.)

Comparison of efficiency of the extraction protocols

DNA quality was estimated by measuring the 260/280 absorbance ratio by a
spectrophotometer (Eppendorf, Beckman D8-800). The success of the extraction
was measured by the DNA concentration, amplification, and yield of amplified
product. Spectral absorbance ratios (A260/280 and, in some cases, A260/230)
correlate with usefulness of DNA for amplification, giving an estimate of the pu-
rity of the solution. However, residual CTAB, RNA contamination, and plant sec-
ondary metabolites can lead to an overestimation of the yield of DNA measured
by the photometer. This method is not useful for small quantities of DNA
(<1 ~tg/mL).
Because densitometric measurements are not useful for the detection of
small amounts of DNA (Csaikl et al., 1998), fluorometric data were used for a
comparison of the quantity of DNA from the different types of molecular extrac-
tions.
Relative concentration was determined by electrophoresing samples after
amplification of the first part of rbcL (RH-1S/Hv362) and trnL-trnF on an
agarose gel (1.3%) and comparing to a standard (Phix 174RF DNA Hae III and
Juncaceae DNA extraction and amplification t 69

pBR 322 DNA/Alw 44 IIMva I). Results from amplification using other rbcL
primers (with a similar length) were approximately identical (data not shown).

Results and Discussion

We summarize methods useful for isolation of cpDNA from herbarium specimens

and compare the results with fresh plants from the same set of samples.
The data show (Table 2, Figures 1-2) that no significant correlation exists
between quality and quantity of amplification products and the age of herbarium
specimens if very specific primers that amplify small product size are used.
Amplification of approximately 300 bp (but not >400 bp) was most effective. The
concentrations of secondary metabolites and polysaccharides vary among tissues
and with tissue age (Ziegenhagen and Scholz, 1998). Extraction techniques solve
this problem. The best extraction from herbarium samples of Juncaceae requires a
good grinding of plant material and a longer precipitation time (results from
Jodrell lab, Kew). The best result at these conditions came from using the DNeasy
Plant Kit (Qiagen).
Effects of preparation of samples and template impurity are greater in her-
barium than in fresh plants. For the purification, the 2 methods used were phenol
extraction and the CsC1 gradient. The phenol extraction has the one disadvantage
that phenol contamination inhibits and reduces the efficiency of downstream reac-
tions, sequencing, and screening (Hiesinger et al., 2001). This is confirmed in
Figure 3, in which a small yield of DNA is documented. Many methods use the
CsCI gradient to remove proteins and polysaccharides. CsC1 gradients are imprac-
tical when milligram and submilligram amounts of tissue specimens are used be-
cause much of DNA might be lost in the gradient (Rogers, 1994). This was
partially confirmed in dry material of Luzula (Table 4). Nevertheless, the amount
of DNA after CsC1 gradient treatment is not always lower than before.
The extraction from leaves of Juncus was not easy. Most Juncus species
(subg. Agathryon = subg. Poiophylli have round or oval blade cross-sections with
slight ridges. Sclerenchyma strands occur just below the epidermis and are scat-
tered in the interior parts of the leaf. Vascular bundles are scattered throughout the
cross section in some taxa (Balsev, 1996). These parts of the leaves are difficult to
grind, containing parenchyma cells that could interfere with the CTAB extraction.
A better situation is in the genus Luzula with linear or lanceolate blades and
in Juncus subg. Juncus, in which a few species with bifacial blades (sect.
Iridifolii, Graminifolii) are found. For this reason, it is necessary to grind the leaf
thoroughly, using the mixer mill. Yields of DNA differ among plant species. In
Juncaceae, it is lower than in many other plant families, and it is difficult to ob-
tain a useful product, especially from herbarium samples.
Table 3 shows a comparison among 4 types of extractions from herbarium
samples. We recommend the DNeasy Plant Kit (Qiagen, not shown in this table,
see Figure 1) and the method described by Doyle and Doyle (1987, 1990), with a
long-term precipitation for extraction. It gave satisfactory results for most sam-
ples, with successful purification and a lower amount of potential contaminants
compared to other techniques. However, the CTAB-based extractions may have
contaminants of CTAB that can lead to an overestimation of the yield in the
170 Dr6bkov6 et al.

Table 2. Yield of extracted DNA after amplification with comparison of age of herbarium specimens.

Concentration DNA
after amplification
Number Taxon Date Age (ng~L) A260/280 A260/230

H205 L. stenophylla 1927 74 24.6 1.53 1.34

H73 L. kjellmaniana 1935 66 28.5 1.55 1.53
H52 L. johnstonii 1936 65 7.6 1.71 2.11
H172 J. himalensis 1952 49 23.3 1.48 1.15
H74 L. kjelmanniana 1952 49 30.2 1.73 2.26
H202 L. subcongesta 19.62 39 26.5 1.51 1.28
H206 L. australasica 1971 30 32.5 1.54 1.46
H199 J. capitatus 1972 29 29.3 1.53 1.41
H196 J. vaginatus 1975 26 23.1 1.38 1.25
H240 J. socotranus 1978 23 38.4 1.55 1.67
H207 L. meridionalis 1979 22 20.7 1.55 1.35
H213 L. africana 1980 21 16.9 1.42 1.35
H201 J. caespititicus 1980 21 16.3 1.51 1.27
H185 L. sylvatica 1983 18 45.2 1.59 1.9
H102 L. giganthea 1989 12 26.0 1.45 1.10
H203 L. novae-cambriae 1994 7 19.4 1.57 1.38
HI06 J. pictus 1996 5 23.2 1.43 1.08
H152 J. polycephalus 1998 3 23.4 1.44 1.10

Figure 1. Amplification of cpDNA rbcL gene using primers RH-1S/Hv362R. (Samples H213, HI06,
Juncaceae DNA extraction and amplification 171
50m

.~ 40

i-
v
<
Z 3O
s
r

t-
O 20

0r

~ 10

' L ' I i I
20 40 60
Age of herbariumspecimens(years)

1.8--

1.7

1.6

A ~tL 9
A
1.5

1.4

1.3
' I ' L ' I 4 I
0 20 40 60 80
Age of herbariumspedrnens(years)
Figure 2. Dependence of DNA concentration after amplification and A260/280 on the, age of
herbarium specimens (no significant correlation between these values at 0.01 significance level,
Spearman coefficient = 0.35 and 0.17, respectively).

photometric measurements, which is caused by interference of residual CTAB in

the samples (Table 3). As mentioned above, the structure of some leaves of
Juncus predispose them for t h e CTAB contamination. For this reason,
172 Drtbkov6 et al.

Figure 3. Amplificationof noncoding cpDNA regions trnL-F using universal primers (Taberletet al.
1991). Protocol 3 was used for herbarium samples and Protocol 8 for fresh samples.

Main isolation problems

One problem with extraction from herbarium specimens is a very low yield of
plant material. Many phylogenetists work with plant taxa that are rare or grown in
inaccessible locations, making it difficult to obtain fresh plant material. The use
of dried plants from historical collections becomes essential for representative
taxonomic sampling. Another problem is the quantity of the suitable tissue avail-
able. Many graminoid plants have a very limited leaf tissue volume (especially
the annual species), and the sampling for a nonproblematic extraction (yielding a
sufficient amount of DNA) would cause serious damage to the herbarium speci-
men. Undoubtedly, especially good homogenization is essential (the best equip-
ment seems to be a mixer mill). Another crucial point is a longer and repeated
precipitation (H. Wittzell, M. Chase, personal communication).
Many protocols for DNA extraction use liquid nitrogen for grinding the
plant material. The homogenization of plant material is easier and faster, but the
simultaneous processing of multiple samples in mortars in one laboratory table
leads to the loss of DNA and contamination of the samples. When PCR products
are analyzed by sequencing, the contamination is revealed. Other techniques, such
as RFLP and RAPD, do not detect this type of mistake.
A good alternative is the use of bead-mills (ceramic cylinders). These cylin-
ders disrupt the plant tissues in microcentrifuges tubes in the mixer mill without
risk of contamination. Insufficient disruption of starting material leads to low
yield and purity. Pulverizing plant material with a mixer mill is easier and pro-
duces DNA of more reliable quality than grinding with liquid nitrogen in a mortar
(Csaikl et al., 1998).
Juncaeeae DNA extraction and amplification 173

"6 ~
~ I'--- I ~ tt~ ~,~ ~-, i ~" t-~

~ 9 . .

~d

N~2
r~

~N,b"
m

,~-MM
o

~ eZ

t"q

~g < < ~

a ~~
9.~ ~
174 Drdbkov6 et aL

Table 4. Comparison of concentration of DNA (ng~l) from herbarium specimens by fluorometry.

QIAquick with QIAquick with long-term

Collection short-term precipitation precipitation and purification
Taxon date in isopropanol (ng~L) in CsCi gradient (ng~L)

L. taiwaniana 1912 4.0 4.9

L. stenophylla 1927 3.8 2.7
L. pumila 1962 7.4 12.1
L. australasica 1972 5.1 6.7
L. meridionalis 1979 3.2 6.3
L. densiflora 1988 12.3 5.9

A final problem is the storage of the extracted DNA. According to Jansen et

al. (1999), DNA isolated from herbarium specimens degrades rapidly. The rate of
degradation is probably no faster than it is for DNA isolated from fresh material.
However, there is often very little high molecular weight DNA to begin with in
isolations from herbarium specimens. Thus, standard storage in TE (10 m m Tris,
1 m m EDTA) buffer at -20~ is not sufficient to preserve the DNA for long peri-
ods. Often, much of the DNA degrades after one year, making it very difficult to
successfully amplify it using PCR. This is the situation with J u n c e a c e a e . The use-
fulness of herbarium extracted DNA decreases very rapidly with time.

Conclusions

The fact that PCR requires only minute amounts of DNA suggests that herbarium
collections will become more valuable as sources of material for molecular phylo-
genetic studies. However, herbarium samples do require special extraction and re-
action conditions. The most crucial points are as follows:
* Proper grinding of plant samples with a mixer mill
9 Long precipitation
9 Use of the DNeasy Plant Kit (Qiagen) for high quality and quantity of DNA
9 Short length of PCR products (optimum of 300-350 bp)
9 Higher number of PCR cycles
9 Short-term storage in TE (_< 1 y)

Acknowledgments

The main part of the experiments were performed in the DNA Laboratory Insti-
tute of Botany ASCR and were supported in part by grants GACR 206/02/0355
and AVOZ6005908. This investigation would not have been possible without the
EU grants: SYS-RESOURCE (Jodrell Laboratory, RBG Kew, UK) and COBICE
(Institute of Botany, University of Copenhagen, DK), where some isolation tech-
niques were tested. L.D. is grateful to Jim Clarson, Jodrell Laboratory RBG Kew,
and Lisbeth Knudsen, Copenhagen University, for technical assistance in the pro-
cess of some isolation and to herbarium curators for the opportunity to collect
samples at K and BM.
Juncaceae DNA extraction and amplification 175

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