全 文 ：Comparison on Four Extraction Methods of Genomic
DNA from Clematis fasciculiflora Franch
HU Yi-chen1，SUN Zheng-hai1* ，WANG Jin1，LI Shi-feng2，XIN Pei-yao1，FAN Xuan1
1． School of Gardening，Southwest Forestry University，Kunming 650224;2． Flower Research Institute，Yunnan Academy of Agricultural
Sciences，Kunming 650205
Abstract ［Objective］This study aimed at comparing the four extraction methods of genomic DNA from Clematis fasciculiflora Franch and deter-
mining the optimal extraction method for extracting the genomic DNA from Clematis fasciculiflora Franch． ［Method］Leavies of Clematis fasciculi-
flora Franch were used as materials for comparing the purity and concentration of extracted DNA and extracting time among the four extraction
methods of genomic DNA including improved CTAB methodⅠ，improved CTAB methodⅡ，improved CTAB methodⅢ and improved SDS method．
［Result］The four extraction methods could all be successfully used for extracting the genomic DNA from Clematis fasciculiflora Franch． The puri-
ty of genomic DNA was the highest using improved CTAB methodⅠ，with the longest extracting time;while the concentration of genomic DNA
was the maximum using the improved SDS method，with the shortest extracting time and relatively low purity;the extracting time of improved
CTAB methodⅢ was the shortest． ［Conclusion］This study had established the optimal extraction method for extracting the genomic DNA from
Clematis fasciculiflora Franch and supported for the further research using molecular biological methods．
Key words Clematis fasciculiflora Franch;Extraction of genomic DNA;Improved CTAB method;Improved SDS method
Received:August 31，2011 Accepted:September 12，2011
Supported by Applied Basic Research Project of Yunnan Province
(2010ZC089) ;the 948 Project of National Forestry Bureau (2008-
4-11) ;Sharing Platform Project of Provincial and Ministerial Key
Subject，Key Laboratory and School Laboratory of Provincial Colle-
ges and Universities in Yunnan Province;Science and Technology
Innovation Fund of Southwest Forestry University．
* Corresponding author． E-mail:sunzhenghai1978@163． com
Clematis belonged to Ranunculaceae，Clematis L．，with a-
bout 355 kinds all over the world［1］ and 147 kinds in China［2 －4］．
Clematis plants were perennial grassy or woody vines，which
were important international ornamental plants［5］ and had a
laudatory title of Queen of Climbing Plant with characteris-
tics such as large flowers，brilliant and abundant colors，vari-
ous flower types，long flowering period and high ornamental
value． Clematis plants had relatively high medicinal value as
well，for instance，the physiological active components they
contained such as triterpenoid saponin and protoanemonin
were medicine inducing diuresis for treating stranguria，dispel-
ling wind and relieving pain，which were commonly used for
clinical and folk application in China［6］． Clematis plants had a
broad market prospect for development and utilization based
on the ornamental and medicinal characteristics．
Clematis fasciculiflora Franch was distributed on grass
slopes，around the pine trees or near the forest in western
Guangxi，western Sichuan and Yunnan of China at the altitude
of 1 000 －2 700 m，which belonged to Clematis L． sect． Chei-
ropsis［7］． Clematis fasciculiflora Franch was winter-blooming
type according to the ornamental characteristics［8］，which was
an important genetic resource of Clematis L． with a long vie-
wing period，resistance to low temperature，high medicinal
value and other advantages． In this study，Clematis fasciculi-
flora Franch were used as materials for the comprehensive
analysis on the effects of the four extraction methods of ge-
nomic DNA，in order to establish the optimal extraction meth-
od for extracting the genomic DNA from Clematis fasciculiflora
Franch and support for the further research using molecular
biological methods．
Materials and Methods
Materials
Clematis fasciculiflora Franch used in this study was col-
lected from the genetic resources field of Flower Institute of
Yunnan Academy of Agricultural Sciences and pretreated as
follows:scrub the leaf surface with 70% alcohol and store in
－70 ℃ refrigerator for use．
Method
Extraction of genomic DNA from Clematis fasciculiflora
Franch Four methods were used to extract the genomic
DNA from Clematis fasciculiflora Franch，including improved
CTAB methodⅠ，improved CTAB methodⅡ，improved CTAB
methodⅢ and improved SDS method，the specific steps were
as follows:
Improved CTAB methodⅠ ① Weigh 0． 5 g of leaves into a
mortar，add liquid nitrogen into the mortar and ground the
leaves into powders，transfer the powders into the 1． 5 ml cen-
trifuge tubes;② Add 0．5 ml of 2 × CTAB (containing β-mer-
captoethanol)buffer into the 1． 5 ml centrifuge tubes，mix
well，keep heated by water bath at 65 ℃ for 60 min，cool to
room temperature;③ Add an equal volume of chloroform-
isoamyl alcohol (24∶1) ，mix well，centrifuge at 12 000 r /min
for 10 min;④ Repeat steps ③ again;⑤ Obtain the superna-
tant，add four volumes of 1 × CTAB precipitation buffer，mix
well，stand for 30 min at room temperature;⑥ Centrifuge at
12 000 r /min for 5 min，obtain the precipitation，and then sus-
pend the precipitation using 400 μl of 1 mol /L Nacl solution;
⑦ Add 2． 5 volumes of absolute alcohol which was stored at
－20 ℃，centrifuge at 12 000 r /min for 5 min;⑧ Rinse the
precipitation using 70% ethanol for twice，blow the precipitati-
on dry and make it dissolved in 100 μl of TE buffer;⑨Add 5 μl
of 10 mg/ml RNaseA，keep heated at 37 ℃ for 60 min，pre-
cipitate using ethanol，make the precipitation dissolved in 100
μl of TE buffer for use．
Improved CTAB method Ⅱ ① －④ steps are in accord-
Agricultural Biotechnology
Agricultural Science ＆ Technology，2011，12(10):1420 －1423
Copyright 2011，Information Institute of HAAS． All rights reserved．
DOI:10.16175/j.cnki.1009-4229.2011.10.019
ance with the improved CTAB method Ⅰ;⑤ Obtain the su-
pernatant，add 2． 5 volumes of absolute alcohol which was
stored at －20 ℃，stand for 60 min at －20 ℃;⑥ Centrifuge
at 12 000 r /min for 5 min，obtain the precipitation;⑦ Rinse
the precipitation using 70% ethanol (volume fraction) for
twice，blow the precipitation dry and make it dissolved in 100
μl of TE buffer．
Improved CTAB method Ⅲ ① Weigh 0． 5 g of leaves into a
mortar，add liquid nitrogen into the mortar and ground the
leaves into powders，transfer the powders into the 1． 5ml cen-
trifuge tubes;② Add 900 μl of 2 × CTAB buffer into the1． 5ml
centrifuge tubes，mix well，keep heated by water bath at 65
℃ for 60 min;③ Cool to room temperature;add 600 μl of
chloroform － isoamyl alcohol (24∶1) ，mix well;④ Centrifuge
at 12 000 r /min for 10 min;obtain the supernatant，add an
equal volume of cold isopropyl alcohol，shake slightly to make
it mix well，stand for more than 30 min at －20 ℃;⑤ Centri-
fuge at 12 000 r /min for 10 min，remove the supernatant;⑥
Rinse the precipitation using 70% ethanol (volume fraction)
for twice，and then rinse the precipitation using absolute alco-
hol for once;⑦ Dry the genomic DNA at room temperature
(at normal temperature for about 20 min) ，add sterile TE so-
lution for the dissolution of DNA，and store at 4 ℃ for use．
Improved SDS method ① Weigh 0． 5 g of leaves into a
mortar，add liquid nitrogen into the mortar and ground the
leaves into powders，transfer the powders into the 1． 5 ml cen-
trifuge tubes，add 1ml of isolation buffer (0． 4 mol /L of glu-
cose，3% of PVP，2% of β-sulfur-based ethanol) ，shake
well，stand for 10 min at room temperature;② Centrifuge at
10 000 r /min for 10 min，remove the supernatant，add 1 ml of
isolation buffer again，centrifuge at 10 000 r /min for 10 min;
③ Remove the supernatant，add 586 μl of SDSfree and 14 μl
of 20% SDS into the precipitation，mix well，keep heated by
water bath at 65 ℃ for 60 min，cool to room temperature，and
then centrifuge at 10 000 r /min for 10 min;④ Obtain the su-
pernatant，add an equal volume of chloroform-isoamyl alcohol
(24∶1) ，mix well slightly for several minutes，centrifuge at
10 000 r /min for 10 min;⑤ Repeat steps ④ again;⑥ Ob-
tain the supernatant，add 2 /3 volume of cold isopropyl alco-
hol，slightly mix well，stand for 30 min in －20 ℃ refrigerator，
centrifuge at 10 000 r /min for 10 min;⑦ Remove the super-
natant，then add 1 ml of 76% ethanol (10 m mol /L NHA4C)
and rinse the precipitation for twice;⑧ Remove the ethanol
carefully，make the genomic DNA naturally dried and then dis-
solved in 100 μl of water with RNase，and store at 4 ℃ for
use．
Determination of genomic DNA of Clematis fasciculiflora
Franch
Determination using Nanovue spectrophotometer The
OD260 /OD280 ratio and concentration of genomic DNA from
Clematis fasciculiflora Franch had been determined using
Nanovue spectrophotometer． Three tubes of genomic DNA
were determined with each extraction method，and each tube
of genomic DNA was determined for three times and
averaged．
Determination through agarose gel electrophoresis The
extraction effects of genomic DNA from Clematis fasciculiflora
Franch with four extraction methods were determined by EB
staining through 1% agarose gel electrophoresis at 200 V．
Three tubes of genomic DNA were determined with each
extraction method．
Results and Analysis
Determination results of genomic DNA of Clematis fascic-
uliflora Franch using Nanovue spectrophotometer
The OD260 /OD280 ratios and concentrations of genomic
DNA from Clematis fasciculiflora Franch were shown in Table
1． OD260 /OD280 ratios of pure plant genomic DNA were usually
ranged between 1． 80 and 1． 90，a OD260 /OD280 ratio larger
than 1． 90 indicated the presence of RNA contamination，and
a OD260 /OD280 ratio less than 1． 60 indicated the presence of
protein or phenol contamination． Accordingly，as can be seen
from Table 1，improved CTAB methodⅠhad shown the best
effect，with OD260 /OD280 ratios ranged between 1． 80 and
1．90． OD260 /OD280 ratios of genomic DNA from Clematis fas-
ciculiflora Franch extracted with improved CTAB method Ⅱ
were larger than 1． 90，suggesting there were relatively high
concentration of RNA． OD260 /OD280 ratios of genomic DNA
from Clematis fasciculiflora Franch extracted with improved
CTAB method Ⅲ and improved SDS method were less than
1． 60，suggesting there were relatively high concentration of
protein or phenol． During the four extraction methods，the im-
proved SDS method had extracted the highest concentration
of genomic DNA with an average of 216． 73 μg /ml，which in-
dicated that there was relatively less DNA loss in the extrac-
tion process with improved SDS method． The concentrations
of genomic DNA extracted with improved CTAB methodⅡand
improved CTAB methodⅢ were close ranging between 161． 51
to 177． 27 μg/ml，and the concentration of genomic DNA ex-
tracted with improved CTAB method Ⅰ was the minimum
(13．58 μg/ml) ，indicating that there was relatively more DNA
loss in the extraction process with improved CTAB methodⅠ．
Determination results of genomic DNA of Clematis fascic-
uliflora Franch through agarose gel electrophoresis
The agarose gel electrophoresis results of the determina-
tion of genomic DNA of Clematis fasciculiflora Franch were
shown in Fig． 1，which revealed that the electrophoresis band
of genomic DNA extracted with improved SDS method was the
brightest，followed by the improved CTAB method Ⅲ，indica-
ting that the concentrations of genomic DNA extracted with
these two methods were relatively higher． However，as can
be seen from the figure，the electrophoresis bands of genomic
DNA extracted with these two methods were divided into two
regions，suggesting the purities of genomic DNA were not
high． During the four extraction methods，the improved CTAB
methodⅠhad shown a dark electrophoresis band with no sig-
nificant regions，which indicated the purity of genomic DNA
was relatively higher with the improved CTAB methodⅠ．
Comparisons on extracting time and complexity of test
procedures among the four extraction methods
Through the comparisons on extracting time and com-
plexity of test procedures among the four extraction methods，
it had been revealed that the extracting time of improved
CTAB methodⅢwas the shortest，as 2． 8 h，and the test pro-
cedure complexity of improved CTAB methodⅢwas the simp-
lest;the extracting time of improved CTAB methodⅠwas the
longest，as 3． 8 h，and the test procedure complexity of im-
proved CTAB methodⅠwas the most complex;the extracting
time of improved CTAB methodⅡ(3．2 h)and improved SDS
method (3．6 h)were close，as well as the complexity of test
procedures．
1241HU Yi-chen et al． Comparison on Four Extraction Methods of Genomic DNA from Clematis fasciculiflora Franch
Table 1 Determination results of genomic DNA of Clematis fasciculiflora Franch using Nanovue spectrophotometer
Method No． OD260 OD280 OD260 /OD280
DNA concentration
μg /ml
Average concentration
μg /ml
Improved CTAB methodⅠ 1 0． 26 0． 14 1． 89 5． 47 13． 58
2 0． 55 0． 31 1． 80 19． 83
3 1． 11 0． 60 1． 85 15． 45
Improved CTAB methodⅡ 1 51． 09 23． 65 2． 16 164． 40 161． 51
2 51． 64 26． 90 1． 92 122． 30
3 56． 15 25． 88 2． 17 197． 80
Improved CTAB methodⅢ 1 18． 73 11． 63 1． 61 256． 80 177． 27
2 8． 68 5． 64 1． 54 137． 80
3 7． 81 4． 97 1． 57 137． 20
Improved SDS method 1 4． 71 2． 80 1． 68 121． 70 216． 73
2 10． 68 6． 36 1． 68 256． 00
3 10． 27 5． 90 1． 74 272． 50
Lane 1 － 3:Improved CTAB methodⅠ;Lane 4 － 6:Improved
CTAB methodⅡ;Lane 7 －9:Improved CTAB methodⅢ;Lane
10 －12:Improved SDS method，M:DL3000 DNA marker．
Fig． 1 Agarose gel electrophoresis results of genomic DNA of
Clematis fasciculiflora Franch extracted with four
extraction methods
Conclusion and Discussion
The four extraction methods had been comprehensively
compared，which had revealed that these four extraction
methods could all be successfully used for extracting the ge-
nomic DNA from Clematis fasciculiflora Franch． To be specif-
ic，the purity of genomic DNA extracted with improved CTAB
methodⅠwas relatively higher，the concentration of RNA was
high in the genomic DNA extracted with improved CTAB meth-
odⅡ，and there were relatively high concentration of protein or
phenol in the genomic DNA extracted with improved CTAB
methodⅢ and improved SDS method． From the perspective
of DNA concentration and the complexity during the extrac-
tion，the concentration of genomic DNA was the minimum u-
sing improved CTAB methodⅠ，with the longest extracting
time;while the concentration of genomic DNA was the maxi-
mum using the improved SDS method，with the shortest ex-
tracting time．
Currently，the common methods for extracting plant ge-
nomic DNA were all evolved from the CTAB method and the
SDS method． Genomic DNA from different plants should be
extracted using corresponding methods，sometimes even the
genomic DNA from different organs in the same plant should
be extracted with different extraction methods，for instance，
some plants had shown better extraction effect using CTAB
method in the leaves，while some were better by using CTAB
method in the seeds［9 －13］． There were two key conditions for
measuring whether a extraction method was ideal or not: (1)
the template DNA should be high in the purity and good in the
integrity with sufficient extraction amount; (2)the extraction
should be simple and rapid to operate with low cost and high
efficiency［14］． However，the two conditions were generally in-
consistent． The more the extracting steps were，the higher
the purity of extracted DNA and the lower the concentration
would be． On the contrary，the lower the purity of genomic
DNA was，the higher the concentration would be，just like im-
proved CTAB methodⅠ and improved SDS method in this
study． Appropriate DNA extraction methods had to be deter-
mined based on the specific research methods and the num-
ber of test materials，considering the different requirements of
the various adopted molecular biological research methods on
DNA．
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Responsible editor:FAN Xiao-hui Responsible proofreader:
檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪
WU Xiao-yan
4 种滑叶铁线莲基因组 DNA提取方法比较(摘要)
胡祎晨1，孙正海1* ，王 锦1，李世峰2，辛培尧1，范 萱1 (1．西南林业大学园林学院，云南昆明 650224;2．云南省农业科学院花
卉研究所，云南昆明 650205)
［目的］对 4种滑叶铁线莲基因组 DNA提取方法进行比较研究，建立滑叶铁线莲最适的 DNA提取方法。
［方法］以滑叶铁线莲叶片为材料，比较改良 CTAB 法Ⅰ、改良 CTAB 法Ⅱ、改良 CTAB 法Ⅲ、改良 SDS法这 4 种基因组 DNA提取法在提取的
DNA纯度、浓度和提取时间等方面的不同。
［结果］4种方法都可提取滑叶铁线莲基因组 DNA。改良 CTAB法Ⅰ提取 DNA纯度最高，但浓度最低且提取时间最长;改良 SDS法提取 DNA
浓度最高，所需时间较短，但纯度较低;改良 CTAB法Ⅲ提取所需时间最短。
［结论］建立了铁线莲最适 DNA提取方法，为运用分子生物学手段对其研究提供支持。
关键词 滑叶铁线莲;基因组 DNA提取;改良 CTAB法;改良 SDS法
基金项目 云南省应用基础研究项目(2010ZC089);国家林业局 948项目(2008-4-11);云南省省部级重点学科、省高校重点实验室及校实验室共享平
台资助项目;西南林业大学科技创新基金。
作者简介 胡祎晨(1989 － )，男，北京大兴人，本科，从事园林植物相关研究，E-mail:342131849@ qq． com。* 通讯作者，副教授，博士，从事园林植物
种质资源评价、采后保鲜及生物技术辅助育种研究，E-mail:sunzhenghai1978@163． com。
收稿日期 2011-08-31 修回日期
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2011-09-12
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青海栽培黄管秦艽的叶绿体 DNA psbA-trnH核苷酸变异和遗传分析(摘要)
张得钧1* ，高庆波2，李福安1，李永平1 (1． 青海大学医学院，青海西宁 810001;2． 中国科学院西北高原生物研究所，青海西宁
810001)
［目的］研究青海栽培黄管秦艽 Gentiana officinalis H． Smith的遗传多样性，对黄管秦艽遗传分化进行分析 ，为其品种选育提供建议。
［方法］应用叶绿体 DNA psbA-trnH序列对青海地区栽培黄管秦艽 6个居群进行遗传多样性分析。
［结果］共发现 10种不同的单倍型，通过单倍型序列间比对，发现了 7个变异位点，黄管秦艽具有较高的遗传多样性(h = 0． 771) ，单倍型多态
性水平(h)为 0． 563 －0． 857，核苷酸多态性水平(π)为 0． 002 43 －0． 006 31。居群遗传分化系数 Gst为 0． 196 0，基因流 Nm 值为 2． 05，80． 40%
遗传变异主要存在于居群内。
［结论］青海栽培黄管秦艽的种质遗传多样性较高 ，有利于培育高品质的药材。
关键词 黄管秦艽;叶绿体 DNA psbA-trnH;遗传变异
基金项目 青海大学中青年科研基金项目(2009-QY-19)。
作者简介 张得钧(1975 －)，男，青海乐都人，副教授，博士，主要从事生药学教学和科研工作，E-mail:djzhang@ nwipb． ac． cn。* 通讯作者。
收稿日期 2011-06-30 修回日期 2011-08-14
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