Please answers these following questions – 1. Read…

Please answers these following questions – 1. Read…

Please answers these following questions:
1. Read the introduction section of the paper. What is its main research area? What new research question does the paper address? Why is this research question important? How does it extend the previous work? 
2. Overall, does the Introduction section make a convincing case for the importance and value of the study? Do the materials and methods provide enough detail for another scientist to repeat the work? Describe why or why not?
3. In the results section, how are the data presented? In pictures, graphs, tables or text? 

For these three questions, it must be answered in complete sentences and a minimum of 2-3 sentences. (but I have 3 or 4 sentences for each)


Fragaria+paper.pdf  Download Attachment

This is an unformatted preview. Please download the attached document for the original format.
A Convenient Protocol for Extraction and Purification of DNA from Fragaria
Author(s): José A. Mercado, Iman El Mansouri, Silvia Jiménez-Bermúdez, Fernando PliegoAlfaro and Miguel A. Quesada
Reviewed work(s):
Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 35, No. 2 (Mar. - Apr., 1999), pp.
152-153
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/4293177 .
Accessed: 11/10/2012 17:38
Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .
http://www.jstor.org/page/info/about/policies/terms.jsp

.
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org.

.

Society for In Vitro Biology is collaborating with JSTOR to digitize, preserve and extend access to In Vitro
Cellular & Developmental Biology. Plant.

http://www.jstor.org

In Vitro Cell. Dev. Biol.-Plant 35:152-153, March-April 1999
C 1999 Society for In Vitro Biology
1054-5476/99
$05.00 + 0.00

A CONVENIENT PROTOCOL FOR EXTRACTION AND PURIFICATION OF DNA
FROM FRAGARIA
FERNANDO LIEGO-ALFARO,t
S
I
A
P
JOSJ? . MERCADO, MANEL MANSOURI, ILVIA IMI?NEZ-BERM1JDEZ,
J
AND IGUEL . QUESADA
M
A
2
d
B
Departamento iologta Vegetal,Universidad e Mdlaga, Campusde Teatinos, 9071 Mdlaga, Spain
(Received 1 December1998; accepted 2 March1999; editor S. Guha-Mukherjee)

SUMMARY

In this paper we describe a simple and efficient DNA extraction protocol for Fragaria species, a highly recalcitrant
genus due to the large amount of polyphenols and polymeric carbohydrates present in strawberry tissues. The protocol
yields a high quality DNA that can be amplified by polymerase chain reaction and digested with restriction endonucleases.
Key words: DNA; Fragaria; strawberry.
w
tumefaciens
F vesca,transformed ith the strain LBA4404of Agrobacterium
carryingthe plasmid pBI121 or the plasmidpBINPLUScontainingan antisense of the pectate lyase gene (MedinaEscobaret al., 1997).
W
Solutionsrequired. e used washingbufferconsistingof 100 mMsodium
acetatebuffer(pH 5), 20 mMEDTA(pH 5), and 0.2 M sorbitol,to which2%
w
PVP (mol.wt. 40 000) and 1% f-mercaptoethanol as addedjust beforeuse;
TE bufferconsistingof 10 mMTris-HCI pH 8.0) and 1 mMEDTA(pH8.0);
(
1
10% N-laurylsarkosine; 0% CTABconsistingof 10% hexadecyltrimethyalcohol(24:1);
b
lammonium romide(wt/vol)in 1 M NaCl;chloroform:isoamyl
phenol saturatedin TE; and ethanol.
DNAextraction rotocol.To extractDNA, we firstgroundtissue into a fine
p
a
powderin liquid nitrogenwith a mortar nd pestle. Washingbuffer(5 ml fbr
0.5 g of sample) was then added and mixed well. After the mixturewas
s
centrifugedat 3000 X g and 4' C for 5 min the supernatant olutionwas
discardedand the pellet was washedwith a similarvolumeof washingbuffer.
Next the pellet was centrifugedat 3000 X g and resuspendedin 3.2 ml of
washingbuffer,to which was added 800 .tl of 5 M NaCl, 500 tl of 10%Na
laurylsarkosine, nd 500 p1of 10% CTAB.The solution was mixed gently
and incubatedat 65' C for 15 min with occasionalshaking. One volumeof
a
chloroform/isoamyllcohol (24:1) was then added and the tube was inverted
until the solution was emulsified. After centrifugingthe solutionat 10 000
s
t
X g, we transferred he supernatant olutionto a new tube and repeatedthe
e
chloroform xtraction.We then added two volumes of 100% cold ethanol,
incubatedthe solution at - 200 C for 15-30 min, centrifugedit at 10 000
X g for5 min, washedthe pellet with80% ethanol,and redissolvedthe pellet
in 400 .tl of TE buffer.If the pellet dissolutionbecame difficult,we heated
it to 65' C. We then added 2 pl of RNase (10 pg/ml) and incubatedthe
solution at 370 C for 30 min to 1 h before extractingit with one volume of
alcohol (25:24:1).This wasfollowedby extraction
phenol/chloroform/isoamyl
a
with chloroform/isoamyl lcohol. Finally, we added two volumes of 100%
ethanol,centrifugedthe solutionat 10 000 X g for 10 min, washedthe pellet
in 80% ethanol, and dried and resuspendedit in 50-100 pl of TE buffer.

INTRODUCTION
Extraction and purification of nucleic acids from many plants are
often difficult due to the high content of interfering compounds present in the extract, mainly polyphenols and polymeric carbohydrates.
These substances bind to the nucleic acids during cell lysis and
cannot be removed by standard methods. In the case of Fragaria
spp., extraction of DNA from fully matured leaves by conventional
methods (Dellaporta et al., 1983; Lassner et al., 1989; Rogers and
Bendich, 1995) often results in a brown, highly viscous solution that
can be neither digested by restriction endonucleases nor amplified
by polymerase chain reaction (PCR). Our current research is focused
on the Agrobacterium-mediated transformation of Fragaria vesca and
Fragaria X ananassa. Lack of a reliable DNA extraction procedure
for these species makes it difficult to select transgenic plants by PCR
and to study the integration of the foreign genes in the genome (El
Mansouri et al., 1996). Manning (1991) reported a procedure for
nucleic acid extraction from strawberry fruits and other recalcitrant
species based on the selective precipitation of carbohydrates and
polyphenols by the solvent 2-butoxyethanol. However, this precipitation is highly dependent on the sodium concentration in the sample
as well as the concentration of the phenol-extracted nucleic acid.
More recently, Porebski et al. (1997) described a modification of the
CTAB extraction method, useful for randomly amplified polymorphic
DNAs (RAPD) analysis in Fragaria spp. In this investigation, we
report an easy and quick protocol that yields a highly purified DNA
from the mature leaves of Fragaria. This DNA can be digested by
restriction endonucleases and amplified by standard PCR. The main
features of this protocol are the partial isolation of the nucleus and
the low pH of the extraction buffer which prevents carbohydrate precipitation and improves the elimination of polyphenols by polyvi-

A
RESULTS NDDISCUSSION
With the method described above, we obtained a colorless DNA
extract, free of the viscous substances that often bind to the DNA
during the ethanol precipitation. The yield varied from 50 to 120 lag
per g fresh weight, with a mean value of 87 ? 22 ltg. This amount
is slightly higher than the yield reported by Porebski et al. (1997)
for this species. In all extractions, the A260/A230 ratios were greater
than 1.8, indicating the absence of contamination with polyphenols
or polysaccharides. Similarly, the A260/A280 ratios varied between

nylpyrrolidone (PVP).
MATERIALSNDMETHODS
A
Plant material. Fully expanded leaves were the source material.These
leaves were obtainedfromplants of Fragaria X ananassa cv. Chandlerand
s
'To whomcorrespondence hould be addressed.

152

EXTRACTION F DNA FROMFRAGARIA
O

m

1

2

3 4.

A

12 3 4 5

7891011

Bu

153

simple method that consisted of removing most of the cytoplasmic
compounds before the rupture of the nucleus, preventing the binding
of these compounds to the DNA. In conclusion, this protocol is a
reliable method to isolate nuclear DNA from strawberry and perhaps
for other recalcitrant species suitable for enzymatic manipulations.
REFERENCES

FIG. 1. A, Electrophoresisof undigested (lanes 1 and 3) and EcoR1digested (lanes 2 and 4) DNA from Fragaria X ananassa (lanes 1 and 2)
and F. vesca (lanes 3 and 4) extractedby the reportedmethod.About 5 ?tg
of DNA were loaded in each well. Line m: molecularweight standard.B,
PCR amplificationof the NPTIIgene in controland transgenicstrawberry
plants. Lane 1: pBI121 plasmid;lane 2: untransformed lant;lane 3: transp
genic F. vesca;lanes 4-7: differentFragaria X ananassaplantstransformed
with the pBI121 vector;lanes 8-11: differentFragaria X ananassa plants
transformed ith the pBINPLUS nti-Pel construct. rrowndicatesthe 255w
A
i
a
a
bp fragmentamplified.The PCR was performed ccordingto Lipp-Joao nd
a
Brown(1993) with 100-500 ng of DNA as template.

1.8 and 2.0, indicating low protein contamination. The purity of the
DNA obtained was confirmed by the complete digestion with restriction endonucleases and by PCR amplification of the gene NPTII (Fig.
1). This protocol has been successfully used to extract DNA from
other strawberry tissues, e.g., mature fruits, and also with micropropagated plants.
Investigators have found precipitation of polyphenolics by PVP to
be critical in obtaining high quality DNA from several plant species
like woody plants and conifers (Kim et al., 1997). The pH is an
important factor that determines the efficiency of polyphenol elimination from vegetal homogenates by PVP. In fact, a low pH facilitates
the formation of hydrogen bonds between plant phenols and PVP
(Andersen and Sowers, 1968). In our case, the use of protocols containing PVP in the extraction buffer failed to eliminate the brown
color typical of phenol contamination. This problem disappeared
when the pH was lowered to 5 in the extraction buffer; this modification has been found to be critical for some RNA extraction protocols (Ainsworth, 1994). In other cases, the suppression of DNA
contamination by carbohydrates has been carried out by different
approaches: enzymatic hydrolysis (Rether et al., 1993), selective precipitation with polyethylene glycol (Li et al., 1994), NaCl/ethanol
(Lodhi et al., 1994) or 2-butoxyethanol (Manning, 1991), and isolation of the nucleus (Wang et al., 1996). In this protocol, we used a

C
Ainsworth, . Isolationof RNA fromfloraltissue of Rumexacetosa(Sorrel).
Plant Mol. Biol. Rep. 12:198-203; 1994.
Andersen, R. A.; Sowers, J. A. Optimumconditions for bonding of plant
7
phenols to insoluble polyvinylpyrrolidone. hytochemistry :293P
301; 1968.
S
version
J
Dellaporta, . L.;Wood, .; Hicks, J. B. A plantDNAminipreparation:
II. Plant Mol. Biol. Rep. 1:19-21; 1983.
El Mansouri, .; Mercado,J. A.; Valpuesta,V.;L6pez-Aranda, . M.; PliegoJ
I
Alfaro, F.; Quesada, M. A. Shoot regenerationand Agrobacteriummediatedtransformationf FragariavescaL. PlantCell Rep. 15:642o
646; 1996.
Kim, C. S.; Lee, C. H.; Shin, J. S.; Chung,Y. S.; Hyung,N. I. A simple and
rapid methodfor isolation of high quality genomic DNA fromfruit
trees and conifers using PVP. Nucleic Acids Res. 25:1085-1086;
1997.
o
Lassner,M. W.;Peterson,P.; Yoder,J. I. Simultaneous mplification f mula
b
tiple DNA fragments y polymerasechain reactionin the analysisof
P
transgenicplantsand theirprogeny. lantMol.Biol. Rep. 7:116-128;
1989.
Li, Q.-B.; Cai, Q.; Guy,C. L. A DNA extractionmethodfor RAPD analysis
from plants rich in soluble polysaccharides.Plant Mol. Biol. Rep.
12:215-220; 1994.
K
o
Lipp-Joao, . H.; Brown,T. A. Enhancedtransformationf tomatoco-cultivatedwithAgrobacterium
C
tumefaciens 58C1Rif:pGSFR1161.Plant
Cell Rep. 12:422-425; 1993.
Lodhi,M. A.; Ye, G.-N.; Weeden,N. F.; Reisch, B. I. A simple and efficient
methodfor DNA extractionfromgrapevinescultivarsand Vitisspecies. Plant Mol. Biol. Rep. 12:6-13; 1994.
Manning,K. Isolation of nucleic acids from plants by differentialsolvent
A
precipitation. nal. Biochem. 195:45-50; 1991.
Medina-Escobar, .; Cardenas,J.; Moyano,E.; Caballero,J. L.; MufiozN
a
c
Blanco,J. Cloning,molecular haracterizationnd expressionpattern
c
of a strawberry-specificDNA with sequence homologyto pectate
lyase fromhigherplants. Plant Mol. Biol. 34:867-877; 1997.
o
Porebski, S.; GrantBailey, L.; Baum, B. R. Modification f a CTABDNA
extractionprotocolfor plants containinghigh polysaccharidesand
polyphenolcomponents.Plant Mol. Biol. Rep. 15:8-15; 1997.
D
Rether, B.; Delmas, G.; Laouedj,A. Isolation of polysaccharide-free NA
fromplants. Plant Mol. Biol. Rep. 11:333-337; 1993.
Rogers, S. O.; Bendich, A. J. Extractionof total cellular DNA fromplants,
algae and fungi. In: Plant Mol. Biol. ManualD1 S. B. Gelvin, R. A.
Schilperoort,eds.; 1-8; Kluwer Academic Publishers, Dordrecht
1995.
f
Wang,X.-D.; Wang,Z.-P.;Zou, Y.-P.An improved rocedure orthe isolation
p
of nuclearDNA fromleaves of wild grapevinesdried with silica gel.
Plant Mol. Biol. Rep. 14:369-373; 1996.