Abstract: A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted in 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses and electrophoresis showed that the DNA extracted using this method was as good and useful as that using standard CTAB method. Key words: DNA extraction; rice; polymerase chain reaction; molecular marker; simple sequence repeats; transgene DNA molecular marker technology has been advancing rapidly during past decades and its application perspective seems very brilliant in crop breeding, varietal purity test of crop seeds and germplasm fingerprinting  MATERIALS AND METHODS Materials Seeds of the following conventional and hybrid rice varieties were used in this study: indica hybrid rice Xieyou 92 (Xieqingzao/Hui 92) F 1 and Xieyou 46 (Xieqingzao A/Milyang 46) F 1 , the cytoplasmic male sterile line Xieqingzao A, the  DNA extraction DNA was extracted using a modified CTAB method  PCR and products analysis PCR amplification of DNA fragments of the cry1Ab gene in transgenic rice and of the microsatellite within the Wx gene in hybrid rice were conducted using the DNA templates isolated by modified CTAB method and our own simplified protocol. The sequences of forward primer and reverse primer for the cry1Ab gene were 5&apos;-TTCCTTGGACGAAATCCCACC-3&apos; and 5&apos;-GCCAGAATTGAACACATGAGCGC-3&apos;, targeting a fragment of 559 bp. The forward primer of SSR of Wx gene was 5&apos;-CTTTGTCTATCTCAAGACAC-3&apos; and the reverse primer 5&apos;-TTGCAGATGTTCTTCCTGATG-3&apos;, which were designed according to Blight et a

Bao Jin-Song

Cui Hai-Rui

Shu Qing-Yao

Wen-Yue Chen

Zhou Xiang-Sheng

CiteSeerX

Rice Science, 2006, 13(1): 67－70                                                                             67 
http://www.ricescience.org 
 
A Simplified Rice DNA Extraction Protocol for PCR Analysis 
CHEN Wen-yue 1, 2, CUI Hai-rui 2, BAO Jin-song 1, ZHOU Xiang-sheng 1, 3, SHU Qing-yao 1 
(1 Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, China; 2 Hangzhou Academy of Agricultural 
Sciences, Hangzhou 310024, China; 3 Zhejiang Seed Administration Station, Hangzhou 310020, China) 
 
Abstract: A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted 
in 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses and electrophoresis showed that the DNA 
extracted using this method was as good and useful as that using standard CTAB method. 
Key words: DNA extraction; rice; polymerase chain reaction; molecular marker; simple sequence repeats; transgene 
 
DNA molecular marker technology has been advancing 
rapidly during past decades and its application perspective 
seems very brilliant in crop breeding, varietal purity test of crop 
seeds and germplasm fingerprinting [1]. Among various DNA 
markers, the PCR-based marker is the most popular and 
efficient one since it needs small amounts of DNA with relative 
low quality [2]. Meanwhile, PCR is also an essential approach 
for detection of target genes in transgenic breeding and genetic 
modified (GM) food detection. Since the DNA extraction 
method was first established by Marmur in 1961 using SDS and 
chloroform, scientists have been devoting themselves to the 
improvement and simplification of DNA extraction methods 
and many kits for DNA isolation are now commercially 
available on the market [3-5]. However, commercial DNA kits, 
though they are very reliable and user-friendly, are too 
expensive to be the choice of researchers working on applied 
sciences, e.g., plant breeding in developing countries. On the 
other hand, most improved or simplified DNA protocols are still 
very tedious and time-consuming when applied to large 
populations in rice breeding programs. Therefore, DNA 
extraction has become a bottleneck for large-scale applications 
of DNA markers and transgenic detection [9]. Based on previous 
reports [10-11], we further simplified the process of DNA 
extraction, and a protocol for rice DNA isolation is reported 
here.  
MATERIALS AND METHODS 
Materials 
Seeds of the following conventional and hybrid rice  
varieties were used in this study: indica hybrid rice Xieyou 92 
(Xieqingzao/Hui 92) F1 and Xieyou 46 (Xieqingzao A/Milyang 
46) F1, the cytoplasmic male sterile line Xieqingzao A, the  
 
Received: 24 October 2005; Accepted: 10 December 2005 
Corresponding author: SHU Qing-yao (qyshu@zju.edu.cn)       
restorer line Milyang 46, and the transgenic japonica rice 
KMD1 (with a cry1Ab gene) and its control variety Xiushui 11. 
DNA extraction 
DNA was extracted using a modified CTAB method [12] 
and a simplified protocol, which was developed in this study. 
Seeds were immersed at 28-30˚C. After germination for 6 days 
in dark, the etiolated seedlings of about 2-3 cm in height were 
collected for DNA isolation. Our simplified protocol included 
the following steps: (1) 10-20 mg etiolated seedlings were cut 
and put into a 1.5-mL eppendorf tube and homogenized using a 
glass pestle in 200 µL of 0.5 mol/L NaOH; (2) 200 µL of 1 
mol/L Tris-HCl (pH=8.0) was added into the tube and mixed 
well; (3) after centrifugation for 10 min at 12 000 r/min, the 
supernatant was used directly for PCR analysis.  
PCR and products analysis 
PCR amplification of DNA fragments of the cry1Ab gene 
in transgenic rice and of the microsatellite within the Wx gene 
in hybrid rice were conducted using the DNA templates isolated 
by modified CTAB method and our own simplified protocol. 
The sequences of forward primer and reverse primer for the 
cry1Ab gene were 5’-TTCCTTGGACGAAATCCCACC-3’ and 
5’-GCCAGAATTGAACACATGAGCGC-3’, targeting a frag- 
ment of 559 bp. The forward primer of SSR of Wx gene was 
5’-CTTTGTCTATCTCAAGACAC-3’ and the reverse primer 
5’-TTGCAGATGTTCTTCCTGATG-3’, which were designed 
according to Blight et al [14]; the forward primer was labeled 
with fluorescent Carboxytetramethylrhodamine when the PCR 
product was analyzed in capillary electrophoresis. The final 
reaction volume was 20 µL, containing 1 µL template DNA, 2.0 
µL 10×PCR buffer, 0.3 µL 10 mmol/L dNTPs, 1.6 µL 25 
mmol/L MgCl2, 1.0 µL 4.0 µmol/L primer (0.4 µmol/L for 
labeled primer), 0.3 U Taq DNA polymerase and ddH2O. The 
PCR was carried out using a PCR Express PX2 (Hybaid). For 
amplification of the cry1Ab gene, DNA was pre-denatured for 4 
min at 94˚C, followed by 30 cycles of reaction: 94˚C for 30 s, 
68                                                                             Rice Science, Vol. 13, No. 1, 2006 
 
54˚C for 60 s and 72˚C for 90 s with a final extension for 7 min 
at 72˚C. For the amplification of SSR of Wx gene, it was done 
as follows: a pre-denature at 94˚C for 4 min, followed by 35 
cycles of 94˚C for 30 s, 55˚C for 60 s and 72˚C for 45 s; and 
finally at 72˚C for 7 min. The reaction without adding template 
DNA was set up as a negative control for PCR analysis every 
time. 
PCR products were separated in agarose gel 
electrophoresis or capillary electrophoresis. In agarose gel 
electrophoresis, the product amplified from cry1Ab gene and 
the SSR from Wx gene were separated by electrophoresis on 
1.5% agarose gel for 1.5 h at 70 voltage and on 3% agrose gel 
for 2 h at 90 voltage in TAE buffer, respectively. A 100 bp DNA 
Ladder Plus (MBI) was used as the standard marker. The gel 
was stained in EB solution and visualized using Gel Doc-1000 
image system (BIO-RAD).  
For capillary electrophoresis, the PCR products were 
purified and denatured before loading. The amplifications in 
tubes were participated at 4℃ for 30 min by adding 1/10 
volume of 7.5 mol/L NH4OAc and 2.5 times volume of ethanol. 
After centrifugation at 12 000 r/min for 30 min, the pellets were 
washed two times with 70% ethanol, dried in air and dissolved 
in 3 µL of ddH2O. Then 0.5 µL of purified DNA solution was 
mixed well with 4.5 µL loading buffer, denatured at 95℃ for 2 
min and then cooled immediately on ice. Capillary gel 
preparation, pre-electrophoresis, sample loading and electro- 
phoresis were carried out according to the operation protocol 
for Mega BACE1000 Sequencer (Amersham Bio Sciences) and 
the data were automatically collected by the equipment. 
RESULTS 
Detection of the transgene  
A DNA fragment of the transgene cry1Ab, with the 
expected size, was amplified through PCR using the DNA 
extracted by both the modified CTAB method and our 
simplified method (Fig. 1). The results indicated that the DNA 
isolated using our protocol could be used as DNA template for 
PCR analysis of transgenes in transgenic plants.  
SSR amplification 
The DNA isolated using the two methods were further 
used for the amplification of the SSR of the Wx gene. Results 
showed that the SSR fragment of Wx gene was amplified using 
DNA extracted by our simplified method and the modified 
CTAB method. A single fragment was amplified from each 
parent, while mixed bands from the hybrids (Fig. 2). Capillary 
electrophoresis also showed that it was useful for documenting 
the amplified fragments from hybrid rice Xieyou 46, where two 
peaks appeared in capillary electropherogram (Fig. 3), which 
implied that the DNA extracted using our simplified protocol 
could also be used for PCR and subsequent analysis using 
capillary electrophoresis. 
DISCUSSION 
DNA markers are of great potential for marker assisted 
selection, germplasm characterization and gene mapping 
because of its advantages over other genetic markers [1, 13, 16] 
During the past decade, significant progress has been made in 
DNA marker development, and consequently, more and more  
abundant and accurate DNA markers are now available for 
various applications. However, the application of DNA markers 
in applied agricultural research, especially in plant breeding, is 
still limited because of cost and effectiveness [17-18]. With regard 
to the whole process of marker development, a simple DNA 
extraction method has become the critical step that needs 
special attention, because the detection and analysis steps are 
 
Fig. 1. Electropherogram of PCR products of cry1Ab fragment. 
Lane M, Marker for molecular weight; Lanes 1 and 3, KMD1 and 
Xiushui 11 amplified from DNA extracted by the simplified method; 
Lanes 2 and 4, KMD1 and Xiushui 11 amplified from DNA extracted 
by CTAB method; Lane 5, Blank control. 
 
 
Fig. 2.  SSR marker electropherogram of Wx gene in rice. 
Lane M, Marker for molecular weight; Lanes 1, 3, 5 and 7,
Xieqingzao A, Milyang 46, Xieyou 46 (F1) and Xieyou 92 (F1) DNA 
extracted by the simplified method, respectively; Lanes 2, 4, 6 and 8, 
Xieqingzao A, Milyang 46, Xieyou 46 (F1) and Xieyou 92 (F1) DNA 
extracted by CTAB method, respectively; Lanes 9 and 10, Xieyou 46, 
DNA stored at -20℃ for 60 days after being extracted by the simplified
and CTAB methods, respectively; Lane 11, Blank control. 
 
CHEN Wen-yue, et al. A Simplified Rice DNA Extraction Protocol for PCR Analysis                                      69 
 
 
now relatively efficient and cost-effective.  
Although various conventional DNA extraction protocols 
are now available, the process is still tedious, time-consuming 
and needs toxic regents [7-8], e.g. CTAB and chloroform are 
needed for CTAB method. Simplification of DNA extraction 
has been a major subject and as a result various methods are 
now available including commercial kits. In rice, the protocol 
reported by Zhang et al [11] and Wang et al [19] were regarded as 
the most simplified and rapid ones. Compared with the 
traditional methods, the simplified protocols or kits are 
convenient or efficient to some degrees, but further 
improvement and simplification are still needed. On the 
principle of alkaline hydrolysis, we developed a very simple 
protocol, which only involves one tube and two steps, in which 
the DNA was extracted only by grinding and centrifuging in 
one tube and could be used as the template in PCR, requiring 
simple facilities and convenient exercise. By this method, the 
extraction of DNA becomes very rapid, and DNA extraction 
could be done for several hundreds of samples per day per 
person, and thus significantly reduces the cost and time for 
DNA isolation (Table 1). 
Transgenic technology and DNA molecular marker 
techniques have become the core of molecular breeding, an 
important research field of plant breeding. Among DNA 
molecular markers, the SSR marker is considered one of the 
excellent genetic markers, because of its co-dominant 
inheritance, relative abundance, high polymorphism, 
reproducibility, and easy analysis [20-21]. Thus the detection of 
transgenes and analysis of SSR markers are rapidly becoming 
important tools in plant variety improvement. We proved that  
the DNA isolated by our simplified protocol could be used as 
DNA template for PCR, and subsequent analysis of agrose gel 
electrophoresis or capillary sequencing electrophoresis. 
Therefore, the protocol should have wide application in DNA 
extraction of other populations for other genes, and thus could 
be useful for high throughput screening of transgenic rice lines, 
marker assisted selection, rapid variety authenticity test and 
germplasm characterization. 
Although the extraction process in our method is very 
simple, attentions still should be paid to avoid DNA degradation, 
e.g. rice seedlings should be ground powerfully and quickly, 
and equal volume Tris-HCl buffer (pH=8) be added 
immediately after the grinding process in NaOH being 
completed. Meanwhile, once the supernatant is transferred to a 
fresh tube and stored at -20˚C, the DNA becomes much stable 
and can be in good quality for several months. 
ACKNOWLEDGEMENTS 
 We are grateful to Mr. Gao Qi-kang, Mao Wei-hua, Shen 
Sheng-quan and Wu Dian-xing for their help in experiments and 
valuable comments on the manuscript. 
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Fig. 3. Capillary electropherogram of SSR Wx in hybrid rice Xieyou 46 from DNA extracted by the simplified method and CTAB method.   
 
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70                                                                             Rice Science, Vol. 13, No. 1, 2006 
 
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A Simplified Rice DNA Extraction Protocol for PCR Analysis

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A Simplified Rice DNA Extraction Protocol for PCR Analysis

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