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DMSO and betaine significantly enhance the PCR amplification of ITS2 DNA barcodes from plants
Journal: Genome
Manuscript ID gen-2019-0221.R1
Manuscript Type: Article
Date Submitted by the Author: 29-Apr-2020
Complete List of Authors: Varadharajan, Bhooma; SRM Institute of Science and Technology, Genetic EngineeringParani, Madasamy; SRM Institute of Science and Technology, Department of Genetic Engineering
Keyword: ITS2 barcode, DMSO, Formamide, Betaine, 7-deaza-dGTP
Is the invited manuscript for consideration in a Special
Issue? :Trends in DNA Barcoding and Metabarcoding 2019
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DMSO and betaine significantly enhance the PCR amplification of ITS2 DNA barcodes from
plants
Varadharajan Bhooma1, Madasamy Parani1*1Genomics Laboratory, Department of Genetic Engineering, SRM Institute of Science and Technology,
Kattankulathur 603 203, Tamil Nadu, India.
Author for correspondence: *[email protected]
Abstract:
ITS2 marker is highly efficient in species discrimination but its application in DNA barcoding is limited
due to huge variations in the PCR success rate. We have hypothesized that higher GC content and the
resultant secondary structures formed during annealing might hinder the PCR amplification of ITS2. To
test this hypothesis, we selected twelve species from 12 different families in which ITS2 was not amplified
under standard PCR reaction conditions. In these samples, DMSO, formamide, betaine, and 7-deaza-dGTP
were evaluated for their ability to improve the PCR success rate. The highest PCR success rate (91.6%)
was observed with 5% DMSO, followed by 1 M betaine (75%), 50 µM 7-deaza-dGTP (33.3%), and 3%
formamide (16.6%). The one sample that did not amplify with DMSO was amplified by adding 1M
betaine. However, combining DMSO and betaine in the same reaction did not improve the PCR.
Therefore, to achieve the highest PCR success rate for ITS2, it is recommended to include 5% DMSO by
default and substitute it with 1 M betaine only in case of failed reactions. When this strategy was tested in
50 species from 43 genera and 29 families, the PCR success rate of ITS2 increased from 42% to 100%.
Keywords: ITS2 barcode, DMSO, Formamide, Betaine, 7-deaza-dGTP
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1. Introduction:
DNA barcoding has been widely used for rapid and accurate species identification. It is also widely used
to authenticate natural herbal materials. Small DNA fragments that are conserved within species and
divergent between species are used as DNA barcode markers. Since this method uses DNA sequences for
identification, it overcomes many of the drawbacks of morphological identification, such as the need to
have intact morphological characters and the difficulty of analysing variations that occur in the
morphology of the plant due to age or geographical location (Gong et al. 2018). For the identification of
animals, the mitochondrial cytochrome c oxidase 1 gene (cox1) is universally used as the standard barcode
(Hebert et al. 2003). This marker was not useful in plant identification as the mitochondrial genomes of
plants have lower evolution rates than those of animals. Maternally inherited chloroplast markers, such as
rbcL, matK, and trnH-psbA, and the biparentally inherited nuclear markers, such as ITS, ITS1, and ITS2,
have been used for species identification in plants (Newmaster, Fazekas and Ragupathy, 2006; Li et al.
2015). Initially, the CBOL plant working group evaluated the PCR recovery, sequence quality, and
discriminatory efficiency of seven chloroplast markers and concluded that a combination of rbcL and
matK can be used as universal core barcodes for DNA barcoding in plants (CBOL, 2009). Later, Chen et
al (2010) identified ITS2 as a novel plant DNA barcode marker that had better discriminatory power and
was more useful in a broader range of plants than rbcL and matK (Chen et al. 2010). In 2011, the China
plant BOL group evaluated rbcL, matK, trnH-psbA and ITS/ITS2 using 6,286 samples and recommended
that ITS2 be used as a core barcode due to its size and ease of handling (Group, China Plant BOL et al.
2011). ITS2 is being used as a core barcode in DNA barcoding of plants for many reasons, including its
smaller size (which makes PCR amplification and bidirectional sequencing full-length DNA barcodes
easier), higher divergence rate, and highly conserved regions, which are useful in phylogenetic studies
(Schultz et al. 2005; Giudicelli et al. 2017).
At the genus level, the PCR amplification rate of ITS2 was found to be relatively higher and it reached as
high as 100% in Sida, Zanthoxylum and Ligusticum (Vassou et al. 2015; Zhao et al. 2018; Liu et al. 2019).
However, there are exceptions, such as the genus Crataegus in which the PCR success rate was only 54%,
even after using additional primer pairs (Zarrei et al. 2015). At the family level, the PCR amplification
rate of ITS2 was relatively lower (85%) in Lamiaceae and Fabaceae (Han and Lin, 2012; Tahir et al.
2018). The lowest PCR success rate of ITS2 (32%) was reported in Lauraceae, and the authors have
concluded that the ITS2 marker isn’t suitable for species identification in this family (Liu et al. 2012). In
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taxonomically diverse groups of plants, the PCR amplification rate of ITS2 reported by different research
groups has varied widely. It was reported to be 75% and 90% in the herbs of Southern Chinese Medicine
and the crude drugs of the Japanese Pharmacopoeia, respectively (Xiaochen et al. 2017; Gong et al. 2018);
however, in a DNA barcoding study on tropical tree species of India, Tripathi et al (Tripathi et al. 2013)
achieved a PCR success rate of only 59%.
The reason why the ITS2 DNA barcode marker isn’t easily amplified in PCR reactions is not clearly
understood. It may be due to the nature of the template, a complete absence of primer binding sites, primers
lacking sufficient homology with primer binding sites, and PCR inhibitors present in the DNA
preparations. Gong et al (2018) suggested that a lack of primer binding or problematic gene structure
might play a significant role in the PCR recovery of ITS2. It has been reported that ITS2 regions from
angiosperms have a high GC content of about 60%, especially in the conserved regions (Hershkovitz and
Zimmer, 1996; Yao et al. 2010). In fact, such conserved regions form secondary structures with specific
patterns, which was found to be useful in phylogenetic studies (Schultz et al. 2005; Giudicelli et al. 2017).
However, such secondary structures, if they remain intact, can potentially stall primer extension, resulting
in PCR failure.
DMSO, betaine, and 7-deaza-dGTP were found to be very effective PCR additives that were useful in the
amplification of the GC-rich target regions with 67% to 79% GC content (Musso et al. 2006). Several
low-molecular-weight amides were also reported to function as PCR enhancers (Chakrabarti and Schutt,
2001). Formamide reduced non-specific amplification and improved the amplification of GC-rich regions
(Sarkar, Kapelner and Sommer, 1990). Glycerol improved the amplification by increasing the specificity
of the PCR (Nagai, Yoshida and Sato, 1998). BSA, PEG, and ammonium sulphate reduced the inhibitory
effects present in the PCR reaction (Liu et al. 2017). In the present study, DMSO, formamide, betaine,
and 7-deaza-dGTP were tested at five concentrations each for their ability to improve the amplification of
ITS2 from the templates in which PCR amplification of ITS2 failed despite several optimization attempts
without PCR additives.
2. Materials & Methods:
Twelve species from twelve different families for which PCR amplification of ITS2 was not successful,
despite several optimization attempts (including varying reaction components and reaction conditions),
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were selected to standardize the PCR amplification using DMSO, formamide, betaine, and 7-deaza-dGTP
as additives (Table 1). Subsequently, the standardized PCR additives were tested in 50 species of
flowering plants representing 43 genera, and 29 families (Table 2).
Genomic DNA was isolated from the plants via a modified cetyl trimethyl ammonium bromide (CTAB)
method (Poovitha et al. 2016). The standard PCR reaction included about 50 ng of genomic DNA as
template, 1X PCR buffer, 0.2mM dNTPs, 5 pmol of forward and reverse primers, and 1U of Taq DNA
polymerase. The ITS2 primers used for PCR amplification were S2F and S3R (Chen et al. 2010).To this
standard reaction mixture, DMSO (1.25%, 2.5%, 5%, 7.5%, or 10%), formamide (1%, 2%, 3%, 4%, or
5%), betaine (0.5 M, 1 M, 1.5 M, 2 M, or 2.5 M), or 7-deaza-dGTP (25 µM, 50 µM, 75 µM, 100 µM or
125 µM) were incorporated to test the efficiency of the PCR additives. A PCR reaction without any
additive was used as a control. All PCR reactions were carried in three replications. The PCR experiments
were carried out in a thermal cycler (Eppendorf, Germany) under the following conditions: initial
denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds,
annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute, followed by a final elongation step
at 72°C for 5 minutes, and then the samples were held at 4°C until they were evaluated by gel
electrophoresis (1% agarose gels).
PCR products were purified by using an EZ-10 spin column PCR purification kit (Bio Basic Inc., Ontario,
Canada). The purified PCR products were checked on 1% agarose gels and subjected to cycle sequencing
reactions using the Big Dye Terminator v3.1 Cycle Sequencing Kit. The samples were bidirectionally
sequenced using a SeqStudio Genetic Analyzer (Thermo Fisher, CA, USA). The sequences were edited
manually and analysed using the NCBI’s Basic Local Alignment Search Tool (BLAST).
Results & Discussions:
DMSO, formamide, betaine, and 7-deaza-dGTP were tested at five different concentrations as additives
in PCR reactions for the amplification of the ITS2 DNA barcode marker from twelve species that
represented 12 families (Figure 1). The PCR success rates in the presence of PCR additives were 92%,
75%, 33%, and 17% for DMSO, betaine, 7-deaza-dGTP, and formamide, respectively. For DMSO, a final
concentration of 5% resulted in the highest PCR success rate (92%) followed by 2.5% (75%), 1.25%
(58%) and 7.5% (58%). For betaine, the 1 M concentration showed the highest PCR success rate (75%),
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followed by 1.5 M (50%) and 0.5 M (41.66%) (Figure 2). To check if DMSO and betaine could
synergistically improve the PCR success rate, betaine (at final concentrations of 0.5 M, 1.0 M, or 1.5 M)
was added to PCR reactions with 5% DMSO. The addition of both betaine and DMSO did not improve
the PCR success rate; in fact, it reduced the PCR success rate when used at the 1.5 M concentration.
Sesamum indicum, Justicia prostrata, Hibiscus panduriformis,and Saccharum officinarum ITS2
sequences were amplified with DMSO but failed to amplify when combined with betaine. However,
though the PCR success rate of 1 M betaine was only 75%, it amplified the one sample that failed to
amplify with DMSO. The one sample that failed with DMSO was Cinnamomum tamala from Lauraceae.
When four other species from Lauraceae were tested, all of them failed to amplify with DMSO but did
amplify with betaine. These results show that there was no synergistic effect between DMSO and betaine,
but also that the two PCR additives can complement each other when used individually. Therefore, it is
recommended that 5% DMSO become part of the standard reaction mixture for the PCR amplification of
ITS2. For samples where ITS2 fails to amplify with DMSO, the DMSO can be replaced with 1 M betaine.
This strategy is desirable economically desirable because betaine is much more expensive than DMSO.
This strategy was tested with a bigger sample size by including 50 taxonomically diverse plant species.
The PCR success rate in the absence of any additives was only 42%, but the addition of 5% DMSO
amplified ITS2 in 92% of the samples and the remaining 8% of the samples were amplified with 1 M
betaine, resulting in a 100% PCR success rate (Table 2). This included four species from Lauraceae that
had a PCR success rate of 32% in the absence of any PCR additives, (Liu et al. 2012) which increased to
only 55% when BSA and DMSO were included as additives (Liu et al. 2017).
It has been reported that GC content of ITS2 may be as high as 80% in plants (Gong et al. 2018). In the
current study, DNA sequencing of the ITS2 regions that were amplified using DMSO and betaine revealed
that the GC content ranged between 61% and 69%. On the other hand, meta-analysis of the nucleotide
composition of the ITS2 DNA barcodes, which were amplified without any PCR additive from 136 species
(representing 66 genera and 31 families) in our previous studies, revealed their GC content to be ranging
between 49% and 67%. A comparison of this data showed that 73% of the samples that amplified in the
presence of an additive had more than 65% GC content, whereas only 4% of the samples that amplified
in the absence of additives had more than 65% GC content (Figure 3). The failure to amplify ITS2 may
be due to ITS2’s high GC content and its distribution in the template DNA. As a result, complementary
DNA strands may fail to separate during denaturation or form intra-strand secondary structures during
A
B
C
D
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annealing. While the former will affect the binding of the primers to the template, the latter will stall the
primer extension beyond the secondary structures. Binding of DMSO to DNA may improve strand
separation and reduce the formation of secondary structures (Kang, Myung and Gorenstein, 2005; Li et
al, 2017), thus leading to enhanced primer annealing as well as extension.
4. Conclusion:
This study and previous reports make it clear that the PCR amplification of ITS2, which is otherwise an
efficient DNA barcoding marker in plants, is greatly affected by ITS2’s higher GC content. The individual
use of DMSO and betaine as PCR additives can enhance the PCR success rate to as high as 100%.
Acknowledgement
We acknowledge the support from SRM-DBT Partnership Platform for Contemporary Research Services
and Skill Development in Advanced Life Sciences Technologies (No.BT/PR12987/INF/22/205/2015).
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Table 1: PCR amplification of ITS2 in the presence/absence of PCR additives
Table 2: The effect of DMSO and betaine on PCR amplification of ITS2 DNA barcodes from
fifty randomly selected species.
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Table 1
S.No. Species
Name Family
PCR amplification of ITS2 in the
presence/absence of PCR additives
No
additive DMSO Formamide Betaine
7-
deaza-
dGTP
1 Crocus
sativus Iridaceae - + - + -
2 Melia
dubia Meliaceae - + - + +
3 Withania
somnifera Solanaceae - + - - -
4 Combretum
albidum Combretaceae - + - - -
5 Albizia
saman Fabaceae - + - + +
6 Justicia
prostrata Acanthaceae - + - + -
7 Cinnamomum
tamala Lauraceae - - - + -
8 Hibiscus
panduriformis Malvaceae - + - + +
9 Aloe
vera Asphodelaceae - + + - -
10 Sesamum
indicum Pedaliaceae - + + + -
11 Saccharum
officinarum Poaceae - + + + +
12 Canna
indica Cannaceae - + - + -
PCR success rate (%) 0 92 17 75 33
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Table 2
S.No. Species name Family PCR amplification of ITS2 barcode marker
Without
DMSO/Betaine
With
5%DMSO
With
1M Betaine
1 Abutilon indicum Malvaceae + + NT
2 Acalypha indica Euphorbiaceae - + NT
3 Alternanthera sessilis Amaranthaceae + + NT
4 Andrographis paniculata Acanthaceae - + NT
5 Asparagus gonoclados Asparagaceae + + NT
6 Azadirachta indica Meliaceae + + NT
7 Canna tropicana Cannaceae + + NT
8 Cardiospermum halicacabum Sapindaceae + + NT
9 Catharanthus roseus Apocynaceae - + NT
10 Centella asiatica Apiaceae - + NT
11 Cinnamomum camphora Lauraceae - - +
12 Cinnamomum macrocarpum Lauraceae - - +
13 Cinnamomum verum Lauraceae - - +
14 Coscinium fenestratum Menispermaceae - + NT
15 Curcuma aromatica Zingiberaceae + + NT
16 Curcuma longa Zingiberaceae - + NT
17 Cynodon dactylon Poaceae + + NT
18 Delonix elata Fabaceae - + NT
19 Diospyros exsculpta Ebenaceae + + NT
20 Eclipta prostrata Asteraceae + + NT
21 Ficus benghalensis Moraceae - + NT
22 Ficus racemosa Moraceae + + NT
23 Ficus religiosa Moraceae + + NT
24 Gymnema sylvestre Apocynaceae - + NT
25 Hibiscus rosa-sinensis Malvaceae - + NT
26 Hybanthus linearifolius Violaceae - + NT
27 Justicia adhatoda Acanthaceae - + NT
28 Litsea glutinosa Lauraceae - - +
29 Melia azedarach Meliaceae - + NT
30 Mimusops elengi Sapotaceae - + NT
31 Moringa oleifera Moringaceae - + NT
32 Ocimum basilicum Lamiaceae - + NT
33 Pavonia zeylanica Malvaceae + + NT
34 Phyla nodiflora Verbenaceae - + NT
35 Phyllanthus niruri Phyllanthaceae + + NT
36 Pongamia pinnata Fabaceae + + NT
37 Salacia reticulata Celastraceae - + NT
38 Saraca asoca Fabaceae - + NT
39 Senna alexandrina Fabaceae + + NT
40 Senna auriculata Fabaceae + + NT
41 Senna italica Fabaceae + + NT
42 Sphaeranthus indicus Asteraceae + + NT
43 Strychnos nux-vomica Loganiaceae + + NT
44 Symplocos racemosa Symplocaceae - + NT
45 Terminalia arjuna Combretaceae - + NT
46 Thespesia populnea Malvaceae - + NT
47 Tribulus terrestris Zygophyllaceae - + NT
48 Typhonium trilobatum Araceae - + NT
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NT = not tested
49 Wrightia tinctoria Apocynaceae + + NT
50 Zingiber officinale Zingiberaceae - + NT
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Figure 1: Effect of the PCR additives DMSO, formamide, betaine, and 7-deaza-dGTP on PCR
amplification of ITS2 DNA barcode markers from twelve species. Addition of DMSO
amplified ITS2 in all the samples, except C. tamala, which was amplified by using betaine.
(M: 100bp DNA marker, NC: negative control without PCR additive).
Figure 2: PCR success rate of ITS2 barcode markers from twelve species in the presence of
different concentrations of DMSO, formamide, betaine, and 7-deaza-dGTP as PCR additive. (-
) indicate PCR success rate in the absence of respective PCR additive.
Figure 3: Comparison of the GC content of the ITS2 DNA barcode marker, which could be
amplified without using any PCR additive (green dots) and in the presence of DMSO or betaine
alone (red dots)
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Figure 1
DMSO Formamide Betaine 7-deaza-dGTP
C.sativus
M.dubia
W.somnifera
C.albidum
A.saman
J.prostrata
C.tamala
H.panduriformis
A.vera
S.indicum
S.officinarum
C.indica
M
NC
1.2
5%
2.5
%
5%
7.5
%
10
%
M
NC
1%
2%
3%
4%
5%
M
NC
0.5
M
1M
1.5
M
2M
2.5
M
M
NC
25
µM
50
µM
75
µM
10
0µ
M
12
5µ
M
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Figure 2
0%
0%
0%
0%
58
.33
%
16
.66
%
41
.66
%
33
.33
%
75
%
16
.66
%
75
%
33
.33
%
91
.60
%
16
.66
%
50
%
25
%
58
.33
%
16
.66
%
0% 1
6.6
6%
50
%
0%
0% 1
6.6
6%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
PCR SUCCESS RATE Vs DIFFERENT ADDITIVE CONCENTRATION
(-)
1.2
5%
2.5
%
5%
7.5
%
10
%
(-)
1%
2%
3%
4%
5%
(-)
0.5
M
1.0
M
1.5
M
2.0
M
2.5
M
(-)
25
µM
50
µM
75
µM
10
0 µ
M
12
5 µ
M
DMSO Formamide Betaine 7-deaza-dGTP
PC
R S
ucc
ess
Rat
e (
%)
Page 16 of 17
https://mc06.manuscriptcentral.com/genome-pubs
Genome
Draft
Figure 3
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120 140 160
GC
%
Sample
65%
Page 17 of 17
https://mc06.manuscriptcentral.com/genome-pubs
Genome