1. Accepted Manuscript
Analytical Methods
Development of An effective and efficient DNA isolation method for Cinna-
momum species
B.S. Bhau, G. Gogoi, D. Baruah, R. Ahmed, G. Hazarika, B. Borah, B. Gogoi,
D.K. Sarmah, S.C. Nath, S.B. Wann
PII: S0308-8146(15)00717-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2015.05.004
Reference: FOCH 17548
To appear in: Food Chemistry
Received Date: 8 January 2015
Revised Date: 28 April 2015
Accepted Date: 1 May 2015
Please cite this article as: Bhau, B.S., Gogoi, G., Baruah, D., Ahmed, R., Hazarika, G., Borah, B., Gogoi, B., Sarmah,
D.K., Nath, S.C., Wann, S.B., Development of An effective and efficient DNA isolation method for
Cinnamomum species, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.05.004
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2. DEVELOPMENT OF AN EFFECTIVE AND EFFICIENT DNA ISOLATION1
METHOD FOR CINNAMOMUM SPECIES2
3
RUNNING TITLE: EFFICIENT DNA ISOLATION FROM CINNAMOMUM4
Bhau BS*, Gogoi G, Baruah D, Ahmed R1
, Hazarika G#
, Borah B, Gogoi B, Sarmah DK,5
Nath SC, Wann SB1
,6
Plant Genomics Laboratory, Medicinal Aromatic & Economic Plants (MAEP) Division,7
CSIR-Northeast Institute of Science & Technology (CSIR-NEIST),8
Jorhat 785006, Assam, India9
1
Biotechnology Division,10
CSIR-Northeast Institute of Science & Technology (CSIR-NEIST),11
Jorhat 785006, Assam, India12
#
Present address - Diagnostic Genetic Lab., Department of Anatomy.13
Assam Medical College (AMC), Dibrugarh, Assam. 785002, India14
*Corresponding Author: Tel.: +91 376 2370121; fax: +91 376 2370011.15
E-mail address: bsbhau@gmail.com; bhaubs@rrljorhat.res.in (BS Bhau)16
17

3. 1
Abstract:18
Different species of Cinnamomum are rich in polysaccharide’s and secondary19
metabolites, which hinder the process of DNA extraction. High quality DNA is the pre-20
requisite for any molecular biology study. In this paper we report a modified method for high21
quality and quantity of DNA extraction from both lyophilized and non-lyophilized leaf22
samples. Protocol reported differs from the CTAB procedure by addition of higher23
concentration of salt and activated charcoal to remove the polysaccharides and polyphenols.24
Wide utility of the modified protocol was proved by DNA extraction from different woody25
species and 4 Cinnamomum species. Therefore, this protocol has also been validated in26
different species of plants containing high levels of polyphenols and polysaccharides. The27
extracted DNA showed perfect amplification when subjected to RAPD, restriction digestion28
and amplification with DNA barcoding primers. The DNA extraction protocol is reproducible29
and can be applied for any plant molecular biology study.30
Key Words: Cinnamomum, DNA isolation, CTAB, activated charcoal, DNA, PCR reaction,31
polyvinyl pyrrolidone32
33

4. 2
1. Introduction34
The Cinnamomum Schaeffer belongs to the family Lauraceae that comprises about35
250 species of trees and shrubs of tropics and sub-tropics (Willis, 1973; Leela 2008).36
Cinnamomum genus (cinnamon) is a very popular spice throughout the world. Out of 2637
species distributed in different parts of India (Hooker, 1885) 12 species are recorded from the38
North-Eastern Part of India (Kanjilal et al., 1940; Deb, 1983; Nath & Baruah, 1984).39
Cinnamon has a broad range of historical uses in different countries of the world and40
different cultures. The species C. zeylanicum Blume originates from Ceylon, being also41
native to Southeast India, are source of cinnamon bark and leaf and their essential oils. Its42
unique qualities are flavor, slightly sweet, pleasant, warm and bitter, besides being strongly43
aromatic (Thakur et al., 1989; Devi et al, 2007; Mir et al, 2004; Rana et al, 2012). Almost44
every part of the cinnamon spice is considered as a remedy for many diseases like45
respiratory, gastritis, menstrual problems, poor circulation, leucorrhea, digestive, diarrhea,46
nausea, gynecological aliments (vaginal yeast infection), dysentery (Chakraborty and Das,47
2010; Begum et al, 2013). Due to economic and ethno-botanical importance world wide, as48
well as, wide variability reported among the species of Cinnamomum, identification based on49
morphological characteristics alone is not sufficient. Cinnamomum needs a thorough insight50
studies that will prove more beneficial to the mankind. In this context, molecular techniques51
as well as several novel DNA-markers (RAPD, RFLP, SSR, ISSR etc.) in the field of biology52
have not only helped us to establish genetic relationship between the members of different53
taxonomic categories but also helped to study close genetic relationship among inter and54
intra-specific genetic variations. DNA based methods also have application in biological55
adulterant detection and authentication of a wide range of food and agricultural commodities56
(Dhanya and Sasikumar, 2010). Identification of true cinnamon from adulterant species based57
on physical traits is very difficult, and the situation is all the more difficult once the58

5. 3
commodity loses its physical form (e.g., powder). Incidentally, powdered bark is more59
frequently used as food flavor and in medicine.60
Plant biochemical profile are immensely influenced by the place of origin, processing61
of the plant tissue; age and tissue type and season (Xie and Leung, 2009; Bressan et al. 2014),62
while place of origin, processing of the plant tissue; age, tissue type and season have no63
influence on the DNA. Chemical markers are further prone to errors, since they need to be64
specific for the species, stable during storage and modification processes and should65
represent therapeutically relevant compound.66
In recent years, although immense progresses in molecular techniques have been67
made but, there are few successful reports available for isolation of pure nucleic acid from68
plant species containing high level of secondary metabolites (Doyle and Doyle 1990;69
Abeysinghe et al. 2009; Sharma and Purohit 2012; Sahu et al. 2012; Swetha et al. 2014).70
These secondary metabolites like polysaccharides and polyphenols severely affect the71
isolation procedure by interacting irreversively with nucleic acid and interfere with the72
function of enzymes in subsequent analysis (Demeke and Adams, 1992, Loomis 1974).73
Polysaccharides, due to their chemical properties, co-precipitate with genomic DNA, giving74
solutions a viscous, glue-like appearance (Porebski et al. 1997) and are known to inhibit75
proper functioning of the enzymes. Phenolics, such as terpenoids and tannins, undergo quick76
oxidation upon their release from leaf tissue and irreversibly bind to the phosphate backbone77
of DNA, characterized by the browning of leaf material (Sahu et al. 2012; Maliyakal, 1992).78
Both contaminants prevent the use of DNA for molecular biology purposes, such as PCR,79
restriction digests, or sequencing by inhibiting the action of polymerases or endonucleases80
(Khanuja et al. 1999). Although there are so many established protocols to isolate nucleic81
DNA from plant tissues, but with high content of secondary metabolites, very few are82
successful and reproducible.83

6. 4
A fast, simple, cost-effective and reliable method is a pre-requisite for any DNA84
extraction and subsequent downstream application. It has been reported that DNA extraction85
from tissues past the budding stage is problematic and also unstable under long-term storage86
(Lodhi et al., 1994). However, in cases when only older tissues are available, a proper87
procedure for selective extraction of DNA is needed. Many protocols have been used in plant88
DNA isolation, but because of the chemical heterogeneity of the species most of them could89
be applied to a limited number of species or even closely related species (Weishing et al.,90
1995). Cinnamomum plant is rich in polyphenols and secondary metabolites, which are major91
hurdles to precipitate purified and high quantity nucleic acids. Another problem that could92
arise during plant DNA isolation is the requirement of liquid nitrogen for crushing the plant93
material as reported in most of the protocols and involvement of lengthy procedure (Ouenzar94
et al., 1998). In the present study most of above-mentioned concerns have been addressed.95
The present investigation is therefore undertaken to standardize a protocol to extract intact96
high quality DNA from Cinnamomum sp. through testing the suitability of the available97
methods and making necessary modification for the purpose.98
2. Material and methods99
Fresh tender leaves of C. impressinervium Meissn., C. zeylanicum Blume and C.100
tamala Nees. & Eberm. were collected in zip lock bags from the pre-identified plots of101
experimental botanical garden of CSIR-NEIST and Assam Agricultural University, Jorhat,102
Assam, India. Keeping in view the criteria for the collection, fresh leaves were collected from103
the similar environmental conditions for DNA isolation studies. For comparing DNA104
concentrations in old and young leaves, the plant material was collected from the same plant.105
The collected leaf material was cleaned with distilled autoclaved water and external moisture106
from the leaves was allowed to air dry. Part of the sample was used fresh and the other half107
was lyophilized for 48 h at -110 °C and then kept in airtight bags till extraction.108

7. 5
2.1. DNA extraction109
For DNA extraction, 400 mg of the lyophilized leaf samples were grounded to110
powder in sterile mortar with silica powder and transferred powdered samples directly to the111
preheated extraction buffer (1.4 M NaCl, 100 mM Tris–HCl [pH 8.0], 20 mM EDTA [pH112
8.0], and 0.2% β-mercaptoethanol, 2% CTAB) and carefully mixed by hand and incubated at113
55 °C for 1 hr. with periodic shaking to avoid aggregation. Another set of experiment was114
conducted parallerly by taking 400 mg of fresh leaf sample and grind the sample to fine115
powder with the help of liquid nitrogen (Table 1). Both the grounded samples were then116
incubated in preheated extraction buffer at 55 °C for 1 hr. The extraction buffer was117
standardised by changing the concentration of one variable at one time i.e. activated charcoal,118
CTAB and Polyvinylpyrrolidone (PVP). Therefore twelve extraction buffers were prepared,119
where 0.5%, 0.7%, 1% and 3% activated charcoal were mixed with 2% CTAB and 2% PVP120
separately, 1%, 2%, 3% and 4% CTAB was mixed with 2% PVP and 0.7% activated charcoal121
separately and 1%, 2%, 3% and 4% PVP was mixed with 2% CTAB and 0.7% activated122
charcoal separately (Table 2). The remaining component of the extraction buffer were kept123
constant i.e. 1.4 M NaCl, 100 mM Tris–HCl [pH 8.0], 20 mM EDTA [pH 8.0], and 0.2% β-124
mercaptoethanol. After incubation of samples in different extraction buffers, the supernatant125
was extracted twice with chloroform: isoamyl-alcohol (24:1 v/v) after centrifugation at126
10,000 rpm at room temperature. To the supernatant extracted pre-chilled ethanol was added127
in equal volume to precipitate the DNA. The sample was centrifuged at 14,000 rpm for 5 min128
and the supernatant was discarded. The DNA pellet was air-dried for 2 h and then suspended129
in 100 µl of 10 mg Ribonuclease A (Sigma R642) in TE/RNase A buffer and incubated at 37130
°C for 30 min. To the incubated solution add equal volume of chloroform-isoamyl alcohol131
(24:1) and centrifuge at 12,000 g for 10 min. DNA was precipitated by the addition of cold132
isopropanol (0.5 Volume) and 100 µl of 2M NaCl to the solution followed by centrifugation133

8. 6
at 10000 rpm at 5 °C. The resulting DNA pellet was washed with 70% ethanol and allowed to134
air dry. The DNA pellet was re-suspended in 50 µL TE buffer and stored at 5 °C for further135
studies.136
To find out the wide applicability of the standardized protocol, 4 (Androgrephis137
paniculata, Litsea cubeba, Azadirachta indica, Cinnamomum camphora) different plant138
species were used for DNA extraction. In addition to this, our standardized DNA extraction139
method was compared with other four conventional methods (Doyle and Doyle 1987; Lee et140
al. 2010; Abeysinghe et al. 2009; Križman et al. 2006) for DNA extraction from C. tamala141
leaves.142
2.2. Quantification of DNA143
The quantity and purity of the extracted DNA was evaluated by using144
biospectrophotometer (Eppendorf, Germany) using an aliquot of 3 µL of DNA sample from145
the stock. The concentration of the extracted DNA was determined using the absorbance at146
260 nm and the purity of the DNA sample was evaluated by the A260/A280 ratio. The purity of147
the DNA bands was also confirmed by 0.8% agarose gel electrophoresis. The bands were148
observed, documented and analyzed using a gel doc system (G: BOX, Syngene, U.K.).149
2.3. Restriction Digestion150
To check the quality of the extracted Cinnamomum DNA, single enzyme restriction151
digestion was also carried out. Single restriction digestion was done using 5 units of EcoR I152
and Hind III (Thermo Scientific, Lithunia) separately. Briefly, the reaction mixture was153
prepared by adding 10 µL of extracted DNA, 15 µL of 2X assay buffer, 10 µL of BSA, and 3154
µL of restriction enzyme (EcoR I and Hind III). Reactions were carried out at 37 °C for 2, 4155
and 6 hrs and the digested products were resolved on 1% agarose gel and visualized through156
Ethidium bromide staining.157
2.4. RAPD Analysis158

9. 7
The DNA quality was confirmed by RAPD technique using decamer primers RPI 12159
(5´ACGGCAACCT 3´) and RPI 15 (5´AGCCTGAGCC 3´) (Bangalore Genei, Bangalore).160
The stock DNA was diluted to 10 ng/µL. The RAPD PCR amplification volume was 25 µL161
containing 1.5 µL of 10X Taq Buffer A with 15 mM MgCl2 (GeNeiTM
), 2.5 mM dNTP162
(Fermentas, Lithunia), 1 µL of Primer (Bangalore Genei), 1.5 µL of 1U/µL Taq DNA163
Polymerase, and the volume was adjusted by adding double distilled autoclaved water. The164
amplification was carried out in a thermal cycler (Veriti® Thermal Cycler, Applied165
Biosystems) using a program configured with a denaturation step of 5 min at 94 °C followed166
by 35 cycles of 30 s at 94 °C, 30 s at 36 °C, and 1 min at 72 °C. The program ended with one167
final extension cycle at 72 °C for 8 min. The amplified product was observed using a gel doc168
system (G: BOX, Syngene, U.K.).169
2.5. DNA Barcoding170
DNA amplification using four sets of DNA barcoding primer for Cinnamomum171
species was done. Four different DNA samples are taken from each of the three different172
Cinnamomum species viz. C. impressinervium, C. zeylanicum and C. tamala. The 4 sets of173
forward and reverse primers were:174
(a) ITS5a-5´-CCTTATCATTTAGAGGAAGGA-3´175
ITS4-5´ -TCCTCCGCTTATTGATATGC-3´) [Kress et al.2005]176
(b) rbcL1F (5´ATGTCACCACAAACAGAAAC-3´)177
–rbcL724r (5´TCGCATGTACCTGCAGTAGC- 3´) [Kress et al.2005]178
(c) rbcL1F (5´-ATGTCACCACAAACAG-3´) &179
rbcL724r (5´-ATGTACCTGCAGTAGC- 3´) [Modified by National Botanical Research180
Institute-NBRI]181

10. 8
(d) trnH (5´-CGCGCATGGTGGATTCACAATCC-3´) –182
psbA (5´GTTATGCATGAACGTAATGCT- 3´)[Kress et al.2005]183
PCR amplification was performed on a Veriti 96 well thermal cycler (Applied184
Biosystems) as follows: 95 °C for 1 min, followed by 35 cycles of 95 °C for 30 s, T °C for 30185
s and 68 °C for 1 min, followed by an elongation step at 68 °C for 5 min. All the PCR186
conditions were the same for all the primer-pairs except the annealing temperature (T) for187
different primer pairs as follows: 53 °C-ITS5a, 53 °C-ITS4, 50 °C for trnH-A, 68 °C for188
psbA, 54 °C for rbcL1F and 58 °C for rbcL-724. Agarose gel (1%) was used for189
electrophoresis of PCR-products. Gel images were obtained using (G: BOX, Syngene, U.K.)190
imaging system.191
3. Results and discussion192
Isolation of high quality of DNA from medicinal and food plant samples is always193
challenging and foremost requirement for molecular biology studies. However, isolation of194
DNA has always not been achievable from many medicinally and economically important195
plants due to the presence of phenolic compounds and secondary metabolites (Sharma and196
Purohit, 2012). Various challenges were encountered during the DNA extraction from197
different accessions of C. tamala, while following the protocol of Doyle and Doyle (1987),198
due to the presence of the high concentration of polysaccharides and phenolic compounds in199
the leaf tissue. The original protocol of Doyle and Doyle and modified CTAB method when200
used did not yield any DNA. Highly viscous, sticky and brownish pellets were difficult to201
handle and colour indicated contamination by phenolic compounds as earlier reported in202
Dimorphandra mollis (Moreira and Oliveira, 2011). The efficiency of the protocol reported203
here was compared against some of the most commonly used plant DNA extraction protocols204
i.e. Doyle and Doyle (1987), Lee et al. (2010), Abeysinghe et al. (2009) and Križman et al.205

11. 9
(2006). These protocols were tested for DNA extraction from non-lyophilized (0.4 gm) and206
lyophilized leaves (0.2 gm) of C. tamala. The DNA isolated was quantified using a207
spectrophotometer at the absorbance of 260 and 280nm. Among different concentrations of208
CTAB (1%, 2%, 3% and 4%), extraction buffer having 2% CTAB gave the best result with a209
DNA yield of 227.56 ± 9.34 ng/mg sample (Table 1) and produced a clear DNA band on the210
agarose gel (Fig. 1a, b). There was no significant difference among fresh or lyophilised leaf211
samples on the quality and quantity of the DNA when used in modified new method (Table212
1). The protocol reported by Abeysinghe et al. (2009) and Lee et al. (2010) yielded very low213
concentration of DNA. Protocol by Križman et al. (2006), which though yielded good214
quantity of DNA, the quality was compromised. The DNA extracted using Križman et al215
(2006) protocol was very viscous and full of mucilage. The Comparative data of DNA216
concentration and absorbance ratio using numerous earlier standardised methods were217
tabulated in table 1. In this communication, few modifications were done to the existing218
protocol of Križman et al (2006) to develop a standardize protocol for high output DNA219
extraction from Cinnamomum sp. Fresh leaf and lyophilized leaf samples yielded similar220
quantity of DNA (Fig.1b and Table 1). This finding was in accordance with the result of221
Chen and Ronald (1999).222
Fresh and young leaf materials are the first choice to obtain good-quality DNA in223
plants (Moreira and Oliveira, 2011). However, mature leaves contain higher quantities of224
polyphenols and polysaccharides, which make it very difficult to isolate DNA of good225
quality. However, even young leaves for the molecular studies is quite challenging for226
species like Cinnamomum. Overcoming this issue using the present optimized protocol227
yielded better quality DNA even from the mature leaf samples. No fragmentation due to228
shearing of DNA during extraction procedure was seen in any of samples and results were229
reproducible. The absence of smears further substantiates the high purity of extracted DNA.230

12. 10
It has been reported previously that shearing of DNA during extraction can directly or231
indirectly interfere with the enzymatic reactions. The average yield of the DNA through this232
modified protocol was found to be 227.56 ± 9.34 ng/µl and the A260/A280 value was found to233
be 1.80 ± 0.09 (Table 1) ensuring that the DNA samples were free from contamination of the234
secondary metabolites and chemicals used during the extraction procedure and were235
amplifiable in PCR reactions. The spectrum of DNA isolated from different species of236
Cinnamomum indicated the ratio of wave of absorbance at wavelength (λ) 260 nm and 280237
nm is 1.82. The gel electrophoresis (0.8% agarose) before RNase treatment indicates the238
presence of impurity (Fig. 1aM), which could be removed after RNase treatment. Picture of239
agarose gel electrophoresis after RNase treatment shows clear intact bands, which proves that240
high molecular weight DNA without degradation was obtained (Fig. 1aL).241
Review and literature suggested the use of 2% CTAB in the extraction buffer as it242
help to disrupt the cell membrane (Saravanaperumal et al., 2012, Bressan et.al. 2014, Doyle243
et al., 1987). By incorporating different concentration of CTAB (e.g., 1%, 2%, 3%, and 4%)244
in the present experiment, 2% CTAB showed better DNA in respect of quality and quantity245
in comparison to other concentration of CTAB. The results showed that at 2% CTAB DNA246
yield was found to be 169.3±13 ng/µl and A260/A280 ratio was 1.81±0.06. Likewise with247
1% CTAB, DNA yield was 85.46±11.88 ng/µl and A260/A280 ratio was 1.38±0.16, with 3%248
CTAB, DNA yield was 169.3±20.43 ng/µl A260/A280 ratio was 1.26±0.12 and 4% CTAB,249
DNA yield was 91.46±3.75 ng/µl A260/A280 ratio was 1.41± 0.05 (Table 2).250
Polyvinylpyrrolidone (PVP) is an important agent to remove the polyphenols by251
forming complex hydrogen bonding with polyphenols and efficiently separate it from DNA252
(Kit and Chandran, 2010). In the extraction buffer, all the components were kept constant and253
PVP concentration was changed to see its effect on the extracted DNA. In our experiment, we254
have used 1%, 2%, 3%, and 4% PVP respectively (Table 2). Khanuja et al., (1999) also used255

13. 11
different concentration of PVP for plants having high content of secondary metabolites like256
polyphenol and polysaccharides. In the present investigation, addition of 2% PVP yielded the257
optimum quality and quantity of DNA. . In our results, approximately 141.73±9.86 ng/µl and258
A260/A280 ratio 1.65 ±0.11was obtained when 2% PVP was used. Likewise at 1% PVP259
produced 92.56 ±6.9 ng/ µl and A260/A280 ratio 1.52±0.09, 3% PVP produces 85.1±5.9 ng/µl260
and A260/A280 ratio 1.32 ±0.04, and at 4% PVP produced 96.1±14.29 ng/ µl and A260/A280261
ratio 1.50 ±0.07 (Table 2). Lade et al. (2013) mentioned that the quality of DNA get declined262
when the PVP concentration was increased and therefore it confirms as our result was263
accurate.264
The intact and high quality of genomic DNA (Fig. 1b, c) obtained could be ascribed265
to the use of a higher concentration of PVP (2.5%) with lower molecular weight (10,000)266
rather than 40,000 (Table 2). A number of workers (Couch and Fritz, 1990; Chaudhry et. al.,267
1999) have recommended the use of PVP with molecular weight of 10,000 at 2% (w/v) to268
address the high concentration of phenolics present in the plant tissue. PVP with low269
molecular weight has less tendency of precipitating with the nucleic acids as compared to270
PVP with high molecular weight thus yielding sufficient amount of polyphenol free DNA271
(Zhang and Stewart, 2000; Križman et al., 2006).272
The principal modification that proved to be fruitful in extraction procedure of DNA273
from Cinnamomum sp. was the use of activated charcoal, high concentration of CTAB274
(2.5%) and precipitation of the DNA under the influence of high salt (2M NaCl). The275
activated charcoal binds with the resinous substances and thereby settles along with the276
debris in the interference layer between the buffer and Chloroform. In addition to PVP,277
activated charcoal also plays a pivotal role as it can absorb resinous matter and coloured278
impurities in the aqueous phase (Bi et al., 1996). Various literatures suggested the use of279
activated charcoal with PVP in the extraction buffer freshly help to remove the polyphenol280

14. 12
more efficiently. Križman et al., (2006) obtained high quality DNA by using 0.5 % activated281
charcoal in the extraction buffer. In the present investigation, 0.7% (w/v) of activated282
charcoal proved to be sufficient in achieving high quality DNA. The incorporation of283
activated charcoal in the extraction buffer before sample get incubated in the water bath284
greatly enhanced the concentration of DNA, and the most appropriate reason for this might285
be by preventing irreversible interaction of DNA with polyphenols since it comes in contact286
with charcoal than DNA (Bi et.al., 1996). In our experiment 0.5% activated charcoal yielded287
148.2±10.9 ng/µl and A260/A280 ratio 1.66 ±0.04, 0.7% activated charcoal yielded 162.8288
±10.35 ng/µl and A260/ A280 ratio 1.83 ±0.05, 1% activated charcoal yielded 122.1±18.68289
ng/µl, and A260/A280 ratio 1.58 ±0.06 and 3% activated charcoal yielded 122±26.09 ng/µl290
and A260/A280 1.44±0.11 ratio (Table 2).291
Wide utility of the protocol was tested by extracting DNA from different plant species292
(Table 3). Using this protocol, DNA was extracted from Androgrephis paniculata, Litsea293
cubeba, Azadirachta indica and Cinnamomum camphora and Cinnamomum tamala. All294
these four species yielded high quality and quantity of DNA (Table 3). Androgrephis295
paniculata, Litsea cubeba, Azadirachta indica, C. camphora and C. tamala yielded296
501.66±76.53 ng/µl, 439.4±18.53 ng/µl, 341.36±30.18 ng/µl, 317.4±25.97 ng/µl and297
568.6±42.73 ng/µl of DNA respectively. This protocol also worked well for different species298
of Cinnamomum (C. tamala, C. impressinervium, C. zeylanicum). Using this protocol good299
quality and quantity of genomic DNA was extracted from all the species under study, which300
are otherwise very difficult species for DNA extraction (Fig. 1c). The DNA extracted from C.301
tamala, C. impressinervium, and C. zeylanicum was 246.82 ng/µl, 190.21 ng/µl and 257.80302
ng/µl respectively.303
Complete digestion with both the restriction enzyme (EcoR1 and Hind III) confirmed304
the purity of DNA (Fig. 2a). High purity DNA is required for PCR and other PCR-based305

15. 13
techniques, such as random amplified polymorphic DNA (RAPD), micro- and macro-satellite306
analyses, restriction fragment length polymorphism (RFLP) and amplified fragment length307
polymorphism (AFLP) used for genome mapping and DNA fingerprinting (Sharma and308
Purohit 2012). The DNA extracted by this method yielded reproducible and scorable bands309
proving its suitability for PCR applications using RAPD, which proves that, there is no310
contamination of PCR inhibitory products (Fig. 2b). The problem that mainly arises in DNA311
extraction are due to the presence of agents like higher contents of polyphenolic compounds,312
resins, latex, Polysaccharides and tannins present in the cell as secondary metabolites usually313
co-precipitate with DNA and interfere with the activity of the DNA polymerase enzyme. The314
presence and the concentration of these compounds vary considerably from plant to plant.315
This protocol is mainly designed for the Cinnamomum sp., which contains very high316
concentration of polysaccharide, but it could be also used for such similar plants successfully.317
Effective DNA barcoding depends on the quality of the biological material. Following318
this simple sampling protocol will ensure proper preservation of biological samples for DNA319
studies. DNA barcoding, using a short gene sequence from a standardized region of the320
genome, is a species identification tool which would not only aid species discovery but would321
also have applications ranging from large-scale biodiversity surveys to identification of a322
single fragment of material in forensic contexts. To fulfill this vision a universal, relatively323
cheap, scalable system needs to be in place. We used rbcL + matK and trnH-psbA primer324
combination to check the quality of Cinnamomum sp DNA isolated using our modified325
protocol (Fig. 2c). All the primer combination and in all the Cinnamomum sp they showed326
the amplification proving the high quality of the DNA isolated. The ITS1 subset produced a327
consistently smaller amplicon with fewer artifactual amplification products and exhibited328
higher levels of sequence divergence relative to ITS2 and was therefore selected for further329
trials against the other loci. A set of 3 different forward and reverse primers for ITS1 were330

16. 14
then evaluated in all possible combinations on the 4 test species, and a consensus primer pair331
was chosen and applied to the entire taxon set for the empirical experiment.332
Based on these findings, it can be concluded that this protocol provides nuclear DNA333
that has little or no visible coloration; possesses a spectrophotometric A260/A280 value >334
1.8, has an intact DNA or at least the mean fragment length more than 10 kb. Moreover, the335
protocol can be used to isolate DNA from young plant leaves as well as younger tissues336
including seedlings, and it works well with frozen tissue, which is suitable in conditions337
when liquid nitrogen is not available. The protocol may also be applied to other medicinal338
plants with mature tissues rich in polysaccharides and polyphenolic compounds.339
Acknowledgements340
The authors are thankful to the Dr. D. Ramaiah, Director, CSIR-North East Institute of341
Science & Technology, Jorhat, Assam, India, for consistent support and encouragement to342
carry out this work and CSIR, Govt. of India, New Delhi for financing the network project343
(BSC-0117). BSB & SCN are thankful to Department of Biotechnology, Government of344
India for research grant under twinning project.345
346

17. 15
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451
452
453

22. 454
455
Fig. 1a. Genomic DNA isolated from plant young leaves (C7, C25, C32) and mature leaves (C7 M, C25456
M, C32M) resolved under 0.8% agarose gel. Lane1 shows the uncut λ DNA457
458
459
460
Figure 1a: Genomic DNA preparation of Cinamomum tamala resolved by electrophoresis461
(0.8% agarose) using the standardize new protocol without RNase treatment (M) and with462
RNase treatment (L):463
(λ: uncut λ DNA, lane 1-8: extract of 8 accessions of Cinamomum tamala)464
465
466
467
Fig 1b Genomic DNA isolated from plant young leaves (C7, C25, C32) and mature leaves (C7 M, C25468
M, C32M) resolved under 0.8% agarose gel. Lane1 shows the uncut λ DNA469
470
471
472
473
474
Fig.1c Genomic DNA of 3 different species of Cinnamomum species C. impressinervium (C-475
24, C-27, C-32, C-55); C. zeylanicum( AAU-A, AAU-B, AAU-C, AAU-7) and C. tamala (476
C-92, C-100, C-101, C-106) resolved by electrophoresis (0.8% agarose) using the standardize477
new protocol . λ: uncut λ DNA.478
479

23. 480
Fig. 2a Restriction digestion of genomic DNA isolated from 3 different species of481
Cinnamomum with EcoR1 (A) and HIND-III(B) for 2hrs (lane 1-3), 4hrs (lane 4-6) and 6hrs482
(lane 7-9) respectively. C.zeylanicum: AAU-6; C. impressinervium: C-32 and C. tamala: C-483
238.484
L is DNA ladder485
486
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9

24. 487
Fig.2b RAPD Profile of DNA isolated from leaf of three different Cinnamomum species viz.488
C. impressinervium (C-24, C-27, C-32, C-55);C. zeylanicum( AAU-A, AAU-B, AAU-C,489
AAU-7) and C. tamala ( C-92, C-100, C-101, C-106) (L-100bp ladder)with primer:(A)RPI-490
12 (B)- RPI-15491
492
493
494
495
496
497
498
Fig. 2c. DNA amplification using Barcoding primer for three different Cinnamomum sp. viz.499
C. impressinervium (C-24, C-27, C-32, C-55) C. Zeylanicum( AAU-A, AAU-B, AAU-C,500
AAU-7) C. tamala ( C-92, C-100, C-101, C-106 ) using (A) ITS5a-ITS4 primer (B) rbcL1F-501
rbcL724r primer (C) rbcL1F- rbcL724r (modified by NBRI) primer (D) trnH- psbA primer502
503
504

25. 23
505
Table 1. Comparrison of diffent DNA isolation methods for yield and purity of the DNA isolated506
using fresh and lypholised leaves of Cinnamomum tamala.507
Average yield of 3 DNA extractions and standard deviation508
Note: - NL: non- lyophilised; L: lyophilised; -ve: negative.509
510
511
512
513
514
515
LANE
METHOD Doyle &
Doyle
Doyle
&
Doyle
Shih-
Chieh
Lee
Shih-
Chieh
Lee
Modified
method
using
charcoal
Modified
method
using
charcoal
Krizma
n
Krizma
n
Abeysing
he
Abeysing
he
Leaf Sample NL L NL L NL L NL L NL L
DNA Concentration
(ng/µl)
-ve -ve 307.3 ±
13.35
334.7 ±
5.9
212.13 ±
7.82
227.56 ±
9.34
174.83
± 10.91
195.1 ±
12.01
315.7 ±
19.97
320.5 ±
19.24
A260/A280 -ve -ve 1.10 ±
0.05
1.12 ±
0.05
1.75 ±
0.09
1.80 ±
0.09
1.57 ±
0.04
1.70 ±
0.11
1.2 ±
0.15
1.17 ±
0.12

26. 24
Table 2. Effect of different concentrations of Charcoal, CTAB and PVP on DNA quantity and quality516
of Cinnamomum tamala517
Average yield of 3 DNA extractions and standard deviation518
519
520
521
LANE
CTAB
concentration
2% 2% 2% 2% 1% 2% 3% 4% 2% 2% 2% 2%
Charcoal
concentration
0.5% 0.7% 1% 3% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7%
PVP
concentration
2% 2% 2% 2% 2% 2% 2% 2% 1% 2% 3% 4%
DNA
concentration
(ng/µl)
148.2
±
10.9
162.8 ±
10.35
122.1±
18.68
122±
26.09
85.46±
11.88
169.3±
13.00
169.3±
20.43
91.46±
3.75
92.56±
6.9
141.73±
9.86
85.1±
5.9
96.1±
14.29
A260/A280 1.66
±
0.04
1.83 ±
0.05
1.58 ±
0.06
1.44±
0.11
1.38±
0.16
1.81±
0.06
1.26±
0.12
1.41±
0.05
1.52±
0.09
1.65±
0.11
1.32±
0.04
1.50±
0.07

27. 25
TABLE 3: The concentration and purity of the DNA samples isolated using modified new method.522
Lane 1 Androgrephis paniculata. Lane 2 cLitsea cubeba. Lane 3 Azadirachta indica. Lane 4523
Cinnamomum camphora. Lane 5 Cinnamomum tamala.524
525
526
Average yield of 3 DNA extractions and standard deviation527
528
529
530
531
LANE
Plant Species Androgrephis
paniculata
Litsea cubeba Azadirachta
indica
Cinnamomum
camphora
Cinnamomum
tamala
DNA
Concentration
(ng/µl)
501.66±76.53 439.4±18.53 341.36±30.18 317.4±25.97 568.6±42.73
A260/A280 1.76±0.07 1.70±0.02 1.64±0.10 1.67±0.11 1.73±0.03

28. 26
High Lights532
• Polysaccharide and secondary metabolites in Cinnamomum hinder DNA extraction.533
• Developed DNA extraction protocol proved better then earlier protocols.534
• An efficient protocol has been developed for extraction of DNA mature leaves of535
Cinnamomum.536
• Extracted DNA was successfully amplified and digested.537
• Extracting DNA from different plant species proved wide utility of the protocol.538
539
540