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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

https://mc06.manuscriptcentral.com/genome-pubs

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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


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


Genome

dmso and betaine significantly enhance the pcr amplification of … · 2020. 6. 24. · sequenced...

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