CTAB-MAKALE

8/6/2019 CTAB-MAKALE

1/18

Plant Molecular Biology Reporter 16: 6986, 1998. 1998 Kluwer Academic Publishers. Printed in Belgium.

Protocols

Comparative Analysis of Different DNA Extraction

Protocols: A Fast, Universal Maxi-Preparation of High

Quality Plant DNA for Genetic Evaluation and

Phylogenetic Studies

U.M. CSAIKL1,, H. BASTIAN2, R. BRETTSCHNEIDER3, S. GAUCH2,A. MEIR2, M. SCHAUERTE4, F. SCHOLZ4, C. SPERISEN5, B.VORNAM6 and B. ZIEGENHAGEN41Austrian Research Centre Seibersdorf, Biotechnology Unit, A-2444 Seibersdorf, Austria;2QIAGEN GmbH, Max-Volmer-Strasse 4, D-40724 Hilden, Germany; 3Centre for Applied

Plant Molecular Biology, AMPII, Institute of General Botany, Ohnhorststrasse 18, D-22609

Hamburg, Germany; 4Federal Research Centre for Forestry and Forest Products, Institute for

Forest Genetics, Sieker Landstrasse 2, D-22927 Grosshansdorf, Germany; 5Swiss Federal

Institute for Forest, Snow and Landscape Research, Zrcherstrasse 111, CH-8903

Birmensdorf, Switzerland;6Institute for Forest Genetics and Plant Breeding, University of

Gttingen, Bsgenweg 2, D-37077 Gttingen, Germany

Abstract. Four DNA extraction protocols were compared for ability to produce DNA fromthe leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials)and rhododendron (silica dried or frozen material). With the exception of maize and poplar,the species are known to be difficult for DNA extraction. Two protocols represented classical

procedures for lysis and purification, and the other two were a combination of classical lysisfollowed by anion exchange chromatography. The DNA obtained from all procedures wasquantified and tested by PCR and Southern hybridisation.Test results indicated superiority ofone of the four protocols; a combination of CTAB lysis followed by anion exchange chro-matography which enabled DNA extraction from all seven species. A second protocol alsoproduced DNA from leaves or needles of all species investigated and was well suited for PCRapplications but not Southern hybridisations. The remaining protocols produced DNA fromsome but not all species tested.

Abbreviations: CTAB, hexadecyltrimethylammonium bromide; EtOH, Ethanol; TBE, tris-borate-EDTA.

Key words: cpDNA, DNA extraction, fingerprinting, forest trees, M13 fingerprinting, method,PCR, rDNA, RFLP, rhododendron, plant

Corresponding author. e-mail: [email protected]; ph: 43 2254 780 3524; fax: 43 2254780 3653.


2/18

70

Introduction

The problem of DNA extraction is still an important issue in the field of

plant molecular biology. Various plants contain high levels of polysaccharidesand many types of secondary metabolites affecting DNA purification. Certainpolysaccharides are known to inhibit RAPD reactions (Pandey et al., 1996).RFLP analysis, cloning, creation of gene banks and various other techniquesare also sensitive to DNA quality. Examples of plants that pose particularproblems with regard to isolation of high quality DNA include rhododendron,oak, and elm. In addition, plant material collected in the field is seldom storedin a manner that ensures DNA quality. For many studies, however, DNA ofhigh quality is essential. Furthermore, projects that involve screening of largenumbers of samples, such as evolutionary or breeding studies, require fastermethods that reliably yield high-quality DNA.

In the course of our project Development and adaptation of rapid mole-

cular screening techniques for assessing genetic diversity in forest trees,isolation of DNA of sufficient amount and quality from forest-tree samplesoften proved to be the limiting step. To circumvent these problems, two newDNA isolation methods were developed by combining the knowledge gainedin the several project groups.

Several laboratories involved in the project performed side-by-side com-parison of all four DNA isolation procedures. Two methods are based onclassical principles of lysis and purification. The first one is the commonlyused protocol of Doyle and Doyle (1990) which has been used successful inmany plant species. The second one, from Guillemaut and Marchal-Drouard(1992), originated from Dellaporta et al. (1983) and was modified according

to Ziegenhagen et al. (1993). The two new protocols combine traditionallysis with anion exchange chromatography for DNA purification (QIAGEN,Hilden, Germany). The methods were evaluated for universality, speed, re-liability, and DNA quality obtained. We evaluated the DNA by performinguniversal PCR-based applications and Southern hybridisations.

Here we report the results of these comparisons and present our solutionfor a new, universal and fast large-scale plant DNA preparation, suitable forvarious genetic studies.


3/18

71

Materials and Methods

Plant material

Two samples each from seven species: Rhododendron luteum (silica driedand frozen), Quercus robur, Ulmus glabra, Abies alba, Pinus sylvestris, Pop-ulus tremula x tremuloides and Zea mays (all fresh) were subjected to DNAextraction. Maize and poplar are easy species for DNA extraction, while theothers (hard species, due to different secondary metabolites) are difficult toextract.

Equipment and chemicals used in all protocols

mixer mill (Type MM2000, 220 V, 50 Hz, Retsch GmbH & Co KG,Haan, Germany) two 25 ml stainless steel buckets and 20 mm stainless

steel beads, centrifuge and rotor capable of 14,000 rpm (= 16, 873 g) and holding

50 ml tubes (e.g. centrifuge no. 2K15, rotor no. 12 139, Sigma Labor-einrichtungen GmbH, Osterode, Germany),

centrifuge and rotor capable of 14,000 rpm (= 12, 929 g) and holding2 ml tubes (e.g. Biofuge 15, Haereus Sepatech, Osterode, Germany),

spectrophotometer DU-7400 (Beckman), Hoechst 33258 dye, luminescence spectrophotometer LS 50 B (Perkin Elmer), WINCAM 2.1 (Cybertech, Berlin, Germany).

Preparation of plant material

For standardisation, all material was homogenised as follows: Two samplesper species were pulverised in a mixer mill for 4 min at 80% max. speedin a stainless steel bucket containing a stainless steel bead (both completelyprecooled in liquid nitrogen). Per sample 1 g of each material (leaves orneedles) was frozen in liquid nitrogen. The frozen powder was immediatelytransferred to 50 ml centrifuge tubes and either stored at 70 C or processedat once.


4/18

72

Testing of DNA extraction protocols

DNA extraction protocol 1 (Carlson/Qiagen)

This new protocol combines a proven lysis procedure for plant DNA isolationand solid-phase technology. DNA isolation with Carlson Lysis Buffer (Carl-son et al., 1991) and QIAGEN Genomic-tips (QIAGEN GmbH, Hilden, Ger-many) was adapted for plant DNA extraction by QIAGEN from the QIAGENblood and cell culture kit.

After lysis and a prepurification step, the lysate is adjusted to conditionsthat allow the DNA to bind to the anion exchange matrix. The DNA immo-bilised on the solid-phase support can be easily purified by removing cellularimpurities using simple washing steps. After washing, DNA is eluted from theanion exchange column, desalted and concentrated by alcohol precipitation.

Chemicals and material

Carlson Lysis Buffer: 2% CTAB, 100 mM Tris/HCl, 1.4 M NaCl, 1%Polyethylenglycol 6000, 20 mM EDTA, pH 9.5

-mercaptoethanol RNase A, 4 mg/ml QIAGEN Genomic tip 500/G (anion exchange column, QIAGEN, Hilden,

Germany) QIAGEN Genomic DNA Buffer Set (QIAGEN, Hilden, Germany) TE pH 8.0 Chloroform/isoamyl alcohol (24:1)

Protocol

To 1 g of ground leaf material add 20 ml of preheated (74 C) CarlsonLysis Buffer followed by 50 l of -mercaptoethanol and 200 l ofRNase A.

Incubate sample for 20 min at 74 C while shaking. Cool to 40 C. Extract the sample once with 20 ml chloroform/isoamyl alcohol (24:1). Add one volume of water to the aqueous phase and adjust pH to 7.0 with

HCl. Load sample onto a QIAGEN Genomic-tip 500/G and purify DNA ac-

cording to the instructions of the manufacturer. Dissolve DNA in 500 l TE.


5/18

73

DNA extraction protocol 2 (Ziegenhagen-upscaled)

This protocol is based on the acidic approach by Guillemaut and Marchal-Drouard (1992) which prevents ionisation and oxidation of polyphenols. DNA

stays soluble while cell debris and contaminants are precipitated. The miniprepversion works well with various woody plant species (Ziegenhagen andFladung, 1997). We increased the leaf material from 100g to 1 g to producean upscaled version of the original acidic DNA extraction protocol describedin Ziegenhagen et al. (1993).

Chemicals

extraction buffer: 100 mM sodium acetate (pH 4.8), 50 mM EDTA (pH8.0), 500 mM NaCl, 2% soluble (w/v) PVP (Sigma, MW 10,000) ad-

justed to pH 5.5 and add SDS to a final of 1.4% (w/v) potassium acetate: 5 M, pH 5.2 10 TE (pH 8.0) phenol phenol/chloroform/isoamyl alcohol, 24:24:1 chloroform/isoamyl alcohol (24:1) ethanol (96%) ethanol (70%) NaOAc: 3 M, pH 5.2 RNase/DNase free (0.5g/l, Boehringer Mannheim, Germany)

Protocol

To 1 g of ground material add 20 ml extraction buffer preheated to 65 C. Incubate samples for 20 min at 65 C with occasionally swirling. Centrifuge for 15 min at 14,000 rpm. To 18 ml of supernatant add 6 ml of potassium acetate and incubate

30 min in an ice bath. Centrifuge for 20 min at 14,000 rpm and split supernatant (28 ml) into

two 15 ml centrifuge tubes. Precipitate DNA by addition of 4.8 ml isopropanol to 8 ml of supernatant

and incubate for 30 min at 20 C. Centrifuge for 15 min at 14,000 rpm at 4 C. Dissolve pellet in 750 l 10X TE and transfer to a 2 ml Eppendorf tube.

Add 10 l RNase and incubate sample for 30 min at 37

C. Perform repeated extractions (each time adding 750l of the solutions

and spin for the time mentioned): 2x phenol (10 min), 2x phenol/chloroform(5 min), 1x chloroform/isoamyl alcohol (10 min).


6/18

74

To 400 l of supernatant add 1100 l ethanol (96%) and 40 l NaOAc,incubate 30 min or overnight at 20 C.

Collect DNA by centrifugation at 14,000 rpm for 30 min (at 4 C).

Wash pellet with 1 ml EtOH (70%) and dissolve DNA in 500 l 1

TE.

DNA extraction protocol 3 (Doyle & Doyle)

As a second classical approach, we used a protocol modified after Doyle andDoyle (1987, 1990) which is the standard DNA preparation used for workwith maize in one of our labs. The protocol is based on lysis and purifica-tion with CTAB which selectively precipitates DNA while maintaining thesolubility of many polysaccharides. CTAB must be washed out carefully.

Chemicals

lysis buffer: 1% CTAB, 1.4 M NaCl, 100 mM Tris/HCl, 20 mM EDTA,

pH 8.0, 2% PVP-40 1% -mercaptoethanol 5 x CTAB solution: 5% CTAB, 0.7 M NaCl chloroform/isoamyl alcohol (24:1) washing buffer: 76% ethanol, 10 mM ammonium acetate TE: 50 mM Tris/HCl, 10 mM EDTA, pH 8.0 RNase solution: 10 mg/ml sodium acetate: 3 M ethanol: (96%)

Protocol

To 1 g of ground leaf material add 10 ml of preheated (65 C) lysisbuffer.

Incubate sample for 30 min at 65 C with occasional swirling. Cool sample to 40 C. Extract the sample once with 10 ml chloroform/isoamyl alcohol. Add 2 ml of 5 x CTAB solution to the aqueous phase. Repeat chloroform/isoamyl alcohol extraction. To precipitate DNA, add 7 ml of isopropanol and incubate the sample at

room temperature for a minimum of 60 min. Centrifuge the DNA at 500 g for 15 min at 4 C. Wash the DNA pellet with 10 ml of washing buffer for 2030 min on a

shaker, swirling gently. Centrifuge the DNA at 3200 g for 10 min at 4 C. Dry the DNA and suspend in 500 l TE1. Add 5 l RNase solution (10 mg/ml) and incubate for 30 min at 37 C.


7/18

75

Repeat phenol/chloroform extraction. Precipitate DNA by addition of 50 l sodium acetate and 1 ml EtOH

and incubate at 4 C for 15 min.

Collect DNA at 13,000 g for 15 min. After washing of the pellet with 70% ethanol, dissolve the DNA in500 l TE.

Notes

1. Store DNA overnight at 4 C to achieve total solution.

DNA extraction protocol 4 (Dellaporta/Qiagen)

This new protocol combines a proven lysis method for plant DNA isolation(Dellaporta et al., 1983) with solid-phase technology (QIAGEN Genomic-tips; QIAGEN GmbH, Hilden, Germany; adapted for plant DNA extractionby QIAGEN from the QIAGEN blood and cell culture kit). After lysis and aprepurification step, the lysate is adjusted to conditions that allow the DNA tobind to the anion exchange matrix. The DNA immobilised on the solid-phasesupport can be easily purified by removing cellular impurities using simplewashing steps. After washing, the DNA is eluted from the anion exchangecolumn, desalted, and concentrated by alcohol precipitation.

Chemicals and materials

Dellaporta Lysis Buffer: 1% SDS, 50 mM Tris/HCl, 100 mM NaCl, 10mM EDTA, pH 8.0

Buffer P3: 3 M potassium acetate pH 5.5 Miracloth (Calbiochem, Bad Soden, Germany)

QIAGEN Genomic tip 500/G (anion exchange column, QIAGEN, Hilden,Germany) QIAGEN Genomic DNA Buffer Set (QIAGEN, Hilden, Germany) TE pH 8.0

Protocol

To 1 g of ground leaf material add 10 ml of preheated (65 C) DellaportaLysis Buffer.

Incubate sample for 15 min at 65 C while shaking. Add 5 ml of ice-cold buffer P3 and incubate the sample for 15 min on

ice.

Remove cell debris and precipitates by centrifugation at 5000 g andfiltration through 2 layers of Miracloth

Load sample onto a QIAGEN Genomic-tip 500/G and purify DNA ac-cording to the instructions of the manufacturer.


8/18

76

Dissolve DNA in 500 l TE.

Comparison of efficiency of different extraction protocols: quantity and

quality of DNA

For DNA quantification, we used spectrophotometer, fluorometer, and densit-ometer readings of band intensity of DNA samples separated on an agarosegels (Table 1). DNA quality was estimated by agarose gel electrophoresis ofgenomic DNA (Figure 1) and application in common downstream protocolsthat would universally detect sequences in all eight species (Figure 24).These protocols included PCR using chloroplast primers (trnS and psbC)(Demesure et al., 1995; Ziegenhagen and Fladung, 1997), and M13 PCRfingerprinting which gives a multi-banding pattern in all plant species andpotentially an individual-specific banding pattern (Degen et al., 1995; Ziegen-

hagen and Fladung, 1997). To test nuclear DNA of all species in one South-ern hybridisation we used a rDNA probe (pTA71) from wheat (Gerlach andBedbrook, 1979).

Results and Discussion

Pulverisation of plant material

Uniformity of the ground plant material is of great importance when extract-ing DNA from hard species. Pulverising plant material with a mixer mill is

easier and produces DNA of more reliable quality than grinding with liquidnitrogen in a mortar. Mechanical disruption of the plant material proved tobe the limiting step when handling multiple samples in parallel. As this stepis the most time consuming part of every plant DNA preparation method,further efforts are needed. A chemical tissue disruption method as used withmammalian cells (Csaikl and Csaikl, unpublished) might be the method ofchoice.

DNA Quality

DNA yield was determined by the three methods described above (Figure 1).

These complex results were assessed by dividing the DNA yields into fourclasses (score 03) as described in Table 1A and summarised in Table 1B.DNA amounts per gram of plant material (means of both extractions perspecies) are scored in four classes as described.


9/18

77

Figure 1. Comparison of DNA yield (g/tissue) obtained by the four methods. DNA wasisolated as indicated from duplicate samples (1, 2) from different species and DNA yield wasdetermined with: OD 260: spectrophotometer reading at 260 nm; fluorometer: reading afterstaining with Hoechst dye 33258; densitometer: scanning band intensity of 1/100 of each

sample separated in parallel on an agarose gel (1.2%, 1

TBE) and stained with ethidiumbromide.


10/18

78

Table 1. Yield of extracted DNA. For estimating contamination, UV-absorbance scansfrom 220-320 nm were run and the DNA yield determined by OD 260 reading. Forfluorometric quantification of DNA with Hoechst dye 33258, the recommended proto-col from Perkin Elmer was used. For densitometric determination of DNA yield, 1/100of each DNA sample was loaded onto a 1.2 % agarose gel and stained with ethidiumbromide. 501000 ng of lambda DNA were used as standard amounts. Scanning of bandintensities was performed using WINCAM 2.1

A. Definition of scores using different measuring systems.

Score Fluorometer Spectrophotometer Densitometer

3 > 100 g/g smooth absorbance scan with symmetricpeak at 260 nm indicating high purity

> 80

2 50100 g/g absorbance scan of DNA is overlaidby absorbance of contaminants at 220240 nm

2080

1 1050 g/g very low absorbance at 260 nm < 200 < 10 g/g no visible peak at 260 nm not visible

B. Scoring of different DNA extraction methods using definitions in A.

Protocol 1 2 3 4

Fluorometer

Spectrophotometer

Densitometer

Fluorometer

Spectrophotometer

Densitometer

Fluorometer

Spectrophotometer

Densitometer

Fluorometer

Spectrophotometer

Densitometer

SpeciesRhododendron luteum 1 3 2 1 3 1 2 1 0 0 0 0

Rhododendron luteum 1 3 2 1 3 1 2 1 0 0 0 0

Quercus robur 3 3 3 2 3 2 1 2 0 0 0 0

Ulmus glabra 1 3 2 1 3 1 3 3 2 3 3 3

Abies alba 3 3 2 2 2 2 1 2 1 0 0 0

Pinus sylvestris 3 3 3 3 2 2 3 3 2 0 0 0

Populus tremula tremuloides 3 3 2 1 3 1 1 3 1 2 3 2

Zea mays 3 3 3 1 2 1 2 3 2 2 2 2

Points 18 24 19 12 21 11 15 18 8 7 8 7

Tot. points / method 61 44 41 22


11/18

79

Comparison of procedures and DNA measurement

In general, there are discrepancies between the DNA measurement methods

due to contaminants and the different properties of the methods. RNA conta-mination, plant secondary metabolites and/or CTAB lead to an overestimationof the yield as assessed by photometric measurements (about 35 higher).Underestimation of DNA yield in densitometric measurements was observedparticularly for high-yield DNA bands and may be explained by saturationwith ethidium bromide (see Figure 1). Additionally, the ability to detect smallamounts of DNA by ethidium bromide staining is limited. Therefore, the fluo-rometric data were used as a basis for comparison between the four moleculartechniques. In spite of this, loading equal (fluorometric measured 2.5 g)amounts of DNA on gels for Southern hybridisation did not always result inloading equally visible quantities (Figure 4B, R. luteum). Protocol 1 enabledextraction of significant amounts of DNA from all species investigated. This

included even the most difficult samples like needles or leaves from variousforest trees or silica-dried leaves of rhododendron. Protocol 2 allows theextraction of sizeable amounts of DNA from all species investigated withexception of rhododendron. The two additional methods under investigationyielded DNA from only some species. Both failed for rhododendron and oak.Protocol 4 additionally failed for the two conifer species (Table 2). Whereverextractions gave sufficient DNA to visualize on agarose gels by ethidiumbromide staining, the DNA also gave a hybridisation signal detectable bySouthern hybridisation (Figure 4 and data not shown).

The classical methods (protocols 2 & 3) are very labour intense andtime-consuming (more than a day). DNA purifications with anion exchange

columns (protocols 1 & 4) speeded up the procedure considerably (3 to 41

2hours total) and result in high yield, and good quality DNA.

DNA Quality

DNA quality was estimated by measuring the 260:280 UV absorbance ratiowhich varied between 1.8 and 2.1. In only a few samples with extremely lowDNA contents was the ratio lower than 1.8 (data not shown).

As DNA quality is of crucial consequence for molecular applications (e.g.Pandey et al., 1996), the DNA was evaluated by performing two PCR basedtechniques and Southern hybridisation. All tests were done with each of the

DNA samples extracted with the four protocols. A detailed description of theresults is presented for protocols 1 & 2 where significant amounts of DNAcould be extracted from all species. Gels and blots are shown in Figures 24. The other methods yield good quality DNA for some species. Protocol 4


12/18

80

R.luteu

s

R.luteu

s,dry

Q.robu

r

U.glabra

A.alba

P.sylve

stris

Populus

var.

Z.may

s

1KBlad

der

1KBladd

er

A

B

Figure 2. Chloroplast trnS-psbCgene region amplification for the seven species investi-

gated. A. Amplification of protocol 1 extracted genomic DNA. B. Amplification of protocol 2extracted genomic DNA Size standard: 1 KB Ladder, Gibco BRL; Primers: organellar/plastidDNA with universal primers representing highly conserved sequences of the 3 regions oftrnS(tRNA-Ser (UGA) and psbCgene (ps II 44kd) Demesure et al. (1995). Primer 1: 5-GGT TCGAAT CCC TCT CTC TC-3; primer 2 (reverse): 5-GGT CGT GAC CAA GAA ACC AC-3;PCR Conditions: Amplification was performed in a 25 l volume of total reaction mixture.The reaction mixture was prepared according to Demesure et al. (1995) and was modified asfollows: 20 ng template DNA, 16.6 mM (NH4)2SO4, 67 mM Tris-HCL, pH 8.2 mM MgCl2,0.001% W1 (Gibco BRL, Life Technologies GmbH), 10 mM -mercaptoethanol, 4.4 g/mlbovine serum albumin, 200 mM of each four dNTP, 1 unit ofTaq polymerase (Gibco BRL,Life Technologies GmbH) and 0.27 M of each primer. Reaction mixtures were kept on iceand covered with 25l of mineral oil. PCR was run in a pre-heated DNA thermal cycler THC1(Perkin-Elmer & Co. GmbH, berlingen, Germany) with the following cycles and tempera-tures (Demesure et al. 1995): 94 C for 4 min, followed by 35 cycles of 93 C for 1 min,57 C for 1 min and 72 C for 2 min. Elongation (72 C) was allowed an additional 10 min.

Visualisation of DNA fragments: PCR products (20 l) were electrophoretically separated on1.0% (w/v) agarose, in Tris-borate buffer at 13 V/cm for 3 hours. DNA fragments were stainedwith ethidium bromide (0.25 g/ml) and visualised by UV fluorescence.


13/18

Table 2. Performance of the DNA isolation methods investigated.

Protocol 1 2 3

Description

Lysis CTAB, pH 9.5 SDS, pH 5.2 CTAB, pH

DNA purification anion exchange phenol/chloroform chloroform

chromatography extraction extraction

Time consumption 4.5 hours > 1 day > 1 day

DNA quantity

Fluorometer 18 12 15

Spectrophotometer 24 21 18

Densitometer 19 11 8

DNA measured in no. of samples 16 16 10

Number of points 77 60 51

DNA quality

PCR amplification of cpDNA 16 16 16

PCR amplification of M13 16 16 16

Performance in Southern/RFLP 16 12 8

Number of points 48 44 40

Tot. number of points 125 104 91

Time required to prepare DNA from 16 pulverised samples.For definition of assignment of points see Table 1.1 point is given for each clear positive result with each sample prepared (16 max.).


14/18

82

gave the overall highest yields for elm. Protocol 3 is, e.g., successfully usedfor routine work with maize.

Both PCR-based applications worked well with DNA from protocols 1

and 2 and resulted in comparable amplification products (Figures 2 and 3).Specific cpDNA amplification of the psbC gene region (about 1,600 bp inlength) was successful for both protocols and all species. The DNA templatefrom protocol 1 produced slightly more PCR products. In a previous investi-gation, this gene region was successfully amplified and subsequently digestedfor 62 angiosperm and gymnosperm tree species (Ziegenhagen and Fladung,1997) using DNA extracted according to the minipreparation procedure ofZiegenhagen et al. (1993). M13 PCR fingerprinting was successful with allsamples. However, DNA bands produced from protocol 1 template were moredistinct. The patterns were reproducible for duplicate DNA preparations fromindividual plants and between individuals of the same species. Only slightmodifications in quantity could be detected. Two oak individuals could be

detected (protocol 2, Figure 3B). Two rhododendron individuals, and twomaize cultivars were extracted in duplicate by protocols 1 and 2 respectively(Figures 3A, B). Furthermore, individual and/or cultivar specific patterns fitresults published for silver fir (Degen et al., 1995).

In Southern analysis, hybridisation signals from the rDNA probe couldbe obtained for all seven sample sets in protocol 1 but no signal was visiblefor rhododendron in protocol 2 (Figures 4A, B). This occurred in spite ofthe fact that according to the fluorometric measurement equal amounts ofgenomic DNA had been loaded. This suggests that the DNA content wasoverestimated due to contaminants in these samples. In general, the hybridi-sation patterns are comparable for the species and duplicates. However, a

gel migration shift between the banding patterns of the oak duplicates fromprotocol 2 was observed. Although the digested DNA from Q. robur appearswell digested (Figure 4B, ethidium bromide stained lanes), the DNA bandsdo not co-migrate completely indicating unequal completion of digestion dueto reduced quality of DNA. The different sized hybridisation signals couldbe mistaken for a polymorphism. Furthermore, different banding patterns arerevealed when the hybridisation patterns of the oak and elm are comparedbetween protocols 1 & 2, most likely due to a partial digestion of the oakDNA extracted with the latter protocol.

Table 2 gives a summary of all methods used to evaluate DNA quantityand quality. Points given for DNA quantity in Table 1 are included. For eval-uation of the DNA quality one point is given for each clear positive result

with each sample tested in the respective application (maximum: 16 points).The PCR-based applications worked for all DNA samples regardless of theDNA extraction method used. Even in those samples where no DNA could


15/18

83

A

B

R.lut

eus

R.lut

eus,dry

Q.robur

U.glabra

A.alb

a

P.sylvestri

s

Popu

lusvar.

Z.ma

ys

1KB

ladde

r

1KB

ladder

Figure 3. M13 PCR Fingerprints for the seven species investigated. A. fingerprints of

protocol 1 genomic extracted DNA. B. Fingerprints of protocol 2 extracted genomic DNA.Size standard: 1 KB ladder, Gibco BRL. Primers: Genomic DNA was amplified with a primerpair from flanking regions of the BsmI/ClaI restriction fragment of the M13mp18 cloningvector (GenBank Accession: # X02513) (Degen et al. 1995). Primer 1 (bp 1768 of M13mp18)5- GTA CTG GTG ACG AAA CTC-3; primer 2 (bp 2531 of M13mp18) 5-ATC GAT AGCAGC ACG GTA-3; PCR Conditions: Amplification was performed in a 25 l volume oftotal reaction mixture (for details see Figure 2) using 0.6 M of each primer (Degen et al.,1995). PCR was run in a pre-heated DNA thermal cycler THC1 (Perkin-Elmer & Co. GmbH,berlingen, Germany) using the following program: 94 C for 4 min, followed by 35 cyclesof 93 C for 1 min, 49 C for 1 min and 72 C for 2 min. Elongation (72 C) was allowedan additional 10 min (Demesure et al. 1995). Visualization of DNA fragments: PCR products(20 l) were electrophoretically separated on a 0.7% (w/v) agarose, in Tris-borate buffer at6 V/cm for 45 min followed by 8 V/cm for 2 h 40 min. DNA fragments were stained withethidium bromide (0.25 g/ml) and visualized by UV fluorescence. The two individuals of

the oak species exhibit a slight modification of pattern.


16/18

84

Hi

ndIII

R.lute

us

R.lute

us,dry

Q.rob

ur

U.glabra

A.alb

aP.

sylves

tris

Popul

usvar

.

Z.mays

R.lute

us

R.lute

us,dry

Q.rob

ur

U.glabra

A.alb

aP.

sylves

tris

Popul

usvar

.

Z.mays

EtBr Hybridisation

HindIII

R.lut

eus

Q.robu

r

U.glabra

A.alb

a

P.sylves

tris

Populus

var.

R.lut

eus

Q.robur

U.glabra

A.alb

a

P.sylves

tris

Popu

lusvar.

Z.ma

ys

Z.ma

ys

B

Figure 4. Southern analysis of EcoR I digested DNA. A. Protocol 1. B. Protocol 2. Hy-bridization was performed for 16 h at room temperature using a rDNA probe pTA71 fromwheat (Gerlach and Bedbrook, 1979). RFLP Analysis of nuclear DNA: rDNA Southern:2.5 g of DNA was digested with restriction enzyme EcoR I and electrophoretically separatedon 0.8% agarose in 1 TAE (40 mM Tris-Acetate pH 7.8, 1 mM EDTA). HindIII digestedphage lambda DNA was used as a size marker. The gels were stained with ethidium bromideand photographed using a Polaroid MP4 camera. DNA was transferred to Amersham HybondN nylon membranes according to the instructions of the manufacturer. Membranes were prehy-bridised for 2 h at room temperature and hybridised to a 32P labelled (Feinberg and Vogelstein,1984) rDNA probe (pTA71 from wheat; Gerlach and Bedbrook, 1979). Hybridization wasperformed for 16 h at room temperature. Prehybridization and hybridization conditions wereas follows: (5 SSPE, 0.1% SDS, 5 Denhardts solution, 30% formamide and 100 g/mlsalmon sperm DNA). Membranes were washed twice at room temperature with 2 SSC, 0.1%

SDS and exposed overnight to X-ray film.


17/18

85

be detected optically, amplification products were obtained (gels not shownfor protocols 3 & 4).

In order to get high quality DNA very fast it is best to use protocol 1 which

also works well with other hard species like heather (results not shown).Nevertheless, we recommend the different protocols be tested for each spe-cific plant of interest. To avoid the occurrence of false polymorphism duringpopulation comparisons especially of hard species we think a cleanup withan anion exchange column is superior to the classical way.

The cost-per-sample varied between the protocols. Protocol 1 (Carlson/QIAGEN) = 34. 38 DM/sample; Protocol 2 (Ziegenhagen upscaled) = 3.97DM/sample; Protocol 3 (Doyle & Doyle) = 4.45 DM/sample and Protocol4 (Dellaporta/QIAGEN) = 30.69 DM/sample. The prices include chemicals,plasticware, and proper disposal of organic solvents in Germany. The price forthe QIAGEN anion exchange columns used in protocols 1 and 4 is 29.50 DMin Germany.

Conclusions

The combination of CTAB lysis followed by anion exchange chromatog-raphy enables a fast and reliable DNA extraction from all species understudy including most difficult tissues like needles or leaves from variousforest trees and silica-dried leaves of rhododendron. Moreover, this DNAis of high quality and yield.

RFLP/Southern analyses is sensitive to DNA quality. To avoid assess-ment of a false positive polymorphism in genetic studies particularlywhen the data are scored by computer programmes, a cleanup of theDNA with anion exchange chromatography is superior to the classicalway.

PCR-based applications are relatively robust against contaminated DNA. Nevertheless, we recommend checking the performance of the different

protocols for a specific plant of choice.

Acknowledgements

This investigation would not have been possible without the EU-Biotechno-logy Program (Project Bio2CT930373). Additionally, the Austrian part of the

project was supported by the Austrian Research Foundation (FWF, project no.P10800-MOB) and the Austrian Ministry for Science and Arts (BMWFK,project no. GZ650.011/2-IV/6/95). We express our gratitude to the Institutefor Forest Genetics (Grosshansdorf, Germany) for hosting the initial practical


18/18

86

Workshop where the topic was discussed and methods compared. We want tothank Dr Roger Hyam (Royal Botanic Garden of Edinburgh) for providingthe rhododendron samples. Our special thanks goes to Drs Kornel Burg and

Franz Csaikl for help with the preparation of the manuscript.

References

Carlson JE, Tulsieram LK, Glaubitz JC, Luk VWK, Kauffeldt C and Rutledge R (1991)Segregation of random amplified DNA markers in F1 progeny of conifers. Theor ApplGenet 83: 194200.

Degen B, Ziegenhagen B, Gillet E and Scholz F (1995) Computer-Aided Search for Codom-inant Markers in Complex Haploid DNA Banding Patterns A Case Study in Abies albaMILL. Silvae Genetica 44: 274-282.

Dellaporta SL, Wood J and Hicks JB (1983) A plant DNA minipreparation: Version II. PlantMol Biol Rep 1: 1921.

Demesure B, Sodzi N and Petit RJ (1995) A set of universal primers for amplification ofpolymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. MolEcol 4: 129131.

Doyle JJ and Doyle JL (1987) A rapid DNA isolation procedure for small quantities of freshleaf tissue. Phytochem Bull 19: 1115.

Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 1315.Feinberg AP and Vogelstein B (1984) A technique for radiolabelling DNA restriction

fragments to high specific activity. Analyt Biochem 137: 266269.Gerlach WL and Bedbrook JR (1979) Cloning and characterization of ribosomal RNA genes

from wheat and barley. Nucleic Acids Res 7: 18691885.Guillemaut P and Marchal-Drouard L (1992) Isolation of plant DNA: A fast, inexpensive and

reliable method. Plant Mol Biol Rep 10: 6065.Pandey RN, Adams RP and Flournoy LE (1996) Inhibition of random amplified polymorphic

DNAs (RAPDs) by plant polysaccharides. Plant Mol Biol Rep 14(1): 1722.

Ziegenhagen B, Guillemaut P and Scholz F (1993) A procedure for mini-preparations ofgenomic DNA from needles of silver fir ( Abies alba Mill.). Plant Mol Biol Rep 11:117121.

Ziegenhagen B and Fladung M (1997) Variation in psbC gene region of gymnosperms andangiosperms as detected by a single restriction site polymorphism. Theor Appl Genet 94:10651071.

Date post:	08-Apr-2018
Category:	Documents
Upload:	cemocanaysun
View:	213 times
Download:	0 times

CTAB-MAKALE

Documents