Optimizing DNA Extraction for Plants with High Polyphenolic Compounds

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Steps to optimize modified cetyltrimethylammonium bromide (CTAB) method for isolation of high quality DNA from plants containing a large amount of mucilage is described. Polymerase chain reaction (PCR) has a wide range of applications in biological studies, genetic, and modern research involving transformations. However, the quantity and quality of template DNA have a significant consequence for reproducible PCR results. Kenaf (Hibiscus cannabinus) contains too much secondary metabolites and polyphenolic compounds, which cause difficulties in extraction of genomic DNA. After failing extracting DNA by three commercial extraction kits, CTAB DNA extraction protocol optimized to extract kenaf genomic DNA extraction. Concentrations of CTAB, PVP, 2-mercaptoethanol (2-ME) and chlorophorm-isoamyl alcohol and duration of incubation in 65 áµ’C have been optimized. Minimal presence of contaminating metabolites revealed by absorbency ratios (A260/A280) in spectrophotometry.Results of this research revealed that 1% CTAB, 0% PVP, 0.5% 2-ME, one time using chlorophorm-isoamyl alcohol mixture and 30 minute incubation in 65 áµ’C were the best treatments. As a conclusion, concentration of 2-mercaptoethanol is the key point to success in extraction of DNA in plants with high mucilage content.

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Introduction

Polymerase chain reaction (PCR) has found vast applications in genetics and molecular biology. However, quality and quantity of base template are one the key points for the reproducible results (Ahmed et al., 2009). Anextraction procedure for every plant often needs to be optimized because the cytoplasm ccontendsare distinctive plant spices. Extraction method may need to be optimized according to growth stage of plant and different organs influences the cytoplasmic contents in plants. Plants containing high levels of polyphenols and polysaccharides posing a major challenge in the isolation of high quality DNA (Moyo et al., 2008). Planteoxyribonucleic acid (DNA) extraction and purification methods have been developed based on Cetrimonium bromide (CTAB), salt extraction (Doyle and Doyle, 1987) and dosodiumodecyl sulfate (SDS)(Mahuku, 2004). Numerous modifications have been reported for the methods (Huang et al., 2000; Michiels et al., 2003; Mahuku, 2004; Savazzini and Martinelli, 2006; Li et al., 2008; Moyo et al., 2008; Ahmed et al., 2009; Ghosh et al., 2009). Every organism in every stage of the growth and different environmental conditions contains various  metabolites and chemical composition so specific extraction buffer  and method would be needed for isolation and purification of DNA. Polyphenolic compounds, polysacarides, secondary metabolites affecting DNA purification.  Some polysaccharides are recognized to hinder RAPD reactions (Pandey et al., 1996). Ghosh, et al., 2009  have reported that use of more volume of extraction buffer and dissolving the crude nucleic acid pellets in 1M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR. Combination of CTAB lysis followed by anion exchange chromatography have been used for DNA extraction by (Csaikl et al., 1998). DNA with a ratio of absorbance (A260/A280) in the range 1.8-2.0 specifies a high level of purity (Pašakinskien and Paplauskien 1999; Weising, 2005)

Materials and methods

Plant material

Young leaf samples from Kenaf, accession 1X51, at flowering stage have been taken in early morning. Leaf samples were transferred to the laboratory on ice and immediately was ground by mortar and pestle in liquid nitrogen. The ground leaf tissue, 0.2 gr, placed in two ml Eppendorf tubes.

Buffres and Readents

List of reagents, their suppliers, and bach numbers have been presented in table 2.

Following mixtures and solutions were used in preparation of extraction buffer and applied during extraction procedure.

0.5 M EDTA stock: 186.12 g of Ethylendiaminetetraacetic acid di-soduium salt (EDTA) was dissolved in 750 ml of H2O. Final pH adjusted to 8.0 with 5 M NaOH

5 M NaCl stock: 292.2 g NaCl added into 700 ml of H2O and the final volume was adjusted to 1.0 l with H2O.

TE buffer: 10ml of 1MTris, pH 8.0, was mixed with 2 ml of 0.5 M sodium EDTA and the final volume adjusted to 1.0 litter with H2O.

Extraction buffer: To make 250ml, 25ml of 1MTris, pH 8.0,70 ml of 5M

NaCl, 10 ml of 0.5 M EDTA and 5 g of CTAB were mixed and the final volume adjusted to

250 ml with H2O. The critical consideration for preparation of extraction buffer is to add 0.5-1% (v/v) of 2-ME to the extraction buffer immediately before use to decrease the possibility of oxidation.

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70% ethanol: Absolute ethanol and H2O were mixed at a ratio of 70 ethanol: 30H2O (v/v).

24:1 chlorophorm-isoamyl alcohol: Chlorophorm and isoamyl alcohol were mixed at a ratio of 24:1 (v/v)

Experimental designs

Experiment 1.

A randomized complete block design with three replications was used. Combinations of PVP in two levels (2% and 3%), 2- merceptoetanol in two levels (3 µl and 6 µl), CTAB in two levels (2 % and 3 %), and application of chlorophorm-isoamyl alcohol in two levels (one and two time applications) were used as atreatments.

Experiment 2.

After conducting first experiment and interpretation of results, second experiment designed in a randomized complete block with three replications and combinations of PVP in two levels (0% and 1%), 2-merceptoetanol in two levels ( µl and 1 µl), CTAB in two levels (0.5 and 1%) and duration of incubation time in 65 áµ’C (30 and 60 minutes) was considered as treatment combinations.

Extraction protocol

Tissue grinding: plant tissue was ground in liquid nitrogen using mortar and pestle. The ground tissue was stored frozen in ultralow freezer until proceeding next step.

200 mg of the ground tissue was added to two ml Eppendorf tubes, subsequently one ml of preheated extraction buffer in a 65 áµ’C added to the frozen tissue powder. The mixture of tissue powder and extraction buffer was mixed thoroughly by 5-10 s vortexes.

The eppendorf tubes, containing buffer and tissue powder, were incubated and mixed in 300 rpm on termomixer comfort for 30 or 60 minutes according to incubation time treatment.

After incubating and mixing, tubes were centrifuged at 13,000 rpm for 10 min at RT to remove non-soluble debris.

800 µl of chloroform:isoamyl alcohol were dispensed to new 2-ml microfuge tubes, then supernatant from previous step were transferred to the microfuge tubes with chloroform:isoamyl alcohol. For taking supernatant, using wide-bore tips is crucial. Afterwards tubes were mixed gently by inverting tubes for 50 times at 20-22 áµ’C (RT).

The phases of the mixture were separate by centrifuging at 13,000 rpm for 10 min at RT in a microfuge.

The aqueous (upper) layer carefully was transferred to a new 2-ml microfuge tube. For the treatments with two time application of chloroform:isoamyl alcohol Five, six and seven steps were repeated.

800 ml cold isopropanol (stored at -20 áµ’C) was added and mixed by inverting the tube and incubated at RT for 10 min to precipitate the DNA.

the mixture was centrifuged at 13,000 rpm for 10 min.

the supernatant was removed using a micropipette and then the pellet air dried.

The pellet washed with 700 µl ethanol then air dried at RT.

Subsequently, pellet was suspended in 50 µl TE at RT.

After complete suspension of pellet 1 µl of DNase-free RNase was added into sample mixture and incubate at 37 áµ’C for 30 min.

Quality and quantity measurements

Quality and quantity of the extracted DNA measured by Nanodrop 2000c. Concentration of DNA (ng/µl) and absorbency ratios (A260/A280) were measured and used for subsequenting analysis of variances.

Results

The results obtained from the analysis of variance of first experiment are presented in Table 1. Significant effect was exposed for the 2-ME, ID, PVP, and CTAB treatments on extracted DNA concentration. Furthermore, some of interaction effects among the treatments were showed significant differences on DNA concentration such as: 2-ME*ID, PVP*ID, PVP*CTAB, 2-ME*ID*PVP, AND 2-ME*ID*CTAB. While, analysis of variances of obtained data from A260/280 of extracted DNA revealed that there are significant differences in levels of ID and PVP treatments. In addition, 2-ME*ID, PVP*ID, 2-ME*CTAB, 2-ME*CTAB*ID, 2-ME*PVP*CTAB, 2-ME*PVP*CTAB*ID treatment interactions were significant.

Table 1. Analysis of variance results for experiment two.

Source of variation

Mean Square

(DNA concentration)

Mean Square

(260A/280A absorbency )

Replication

338046.4*

0.0045**

2-MEa

13073073.1**

0.0017n.s

IDb

2242902.1**

0.0230**

2-ME*ID

3348946.5**

0.0028*

PVPc

4756565.0**

0.0032*

2-ME*PVP

202501.1n.s

0.0001n.s

PVP*ID

1305447.3**

0.0032*

2-ME*PVP*ID

679561.4*

0.0006n.s

CTABd

506.3*

0.0002n.s

2-ME*CTAB

403755.1n.s

0.0054**

CTAB*ID

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28523.6n.s

0.0003n.s

2-ME*CTAB*ID

4413682.5**

0.0063**

PVP*CTAB

3490511.4**

0.0006n.s

2-ME*PVP*CTAB

132520.56n.s

0.0050**

PVP*CTAB*ID

98908.4n.s

0.0009n.s

2-ME*PVP*CTAB*ID

126516.1n.s

0.0028*

Coefficient of Variation

8.2%

1.23%

n.s. Non-significant

* Significant in 5%

** Significant in 1%

a 2-Mercaptoethanol

b Incubation duratuon

c Polyvinylpyrrolidone

d Hexadecyltrimethylammonium bromide

Table 2 provides the extracted DNA concentration and A260/280 absorbency means and standard deviations for different levels of treatments.

Table 2. Means of treatments for Experiment 1.

DNA concentration ng/µl

260A/280A absorbency ratio

Treatment

Mean

Std. Dev.

Mean

Std. Dev.

2-MEa0

3342.2

768.5

1.978

0.045

2-ME 1

4386.0

701.7

1.966

0.039

PVPb 0%

4178.9

850.5

1.980

0.049

PVP 1%

3549.3

849.2

1.964

0.032

CTABc0.5%

3867.3

537.7

1.970

0.045

CTAB 1%

3860.8

1167.2

1.974

0.040

IDd30 min

3647.9

1143.7

1.994

0.046

ID 60 min

4080.2

495.8

1.950

0.022

a 2-Mercaptoethanol

b Polyvinylpyrrolidone

c Hexadecyltrimethylammonium bromide

d Incubation duratuon

The most concentrated DNA obtained from ID of 30 minutes with 4386 ng/µl, while lowest concentration observed for %0 2-ME with 3342.2 ng/µl. Absorbency ratio A260/280 ranged from 1.950 to 1.994 for ID 60 minutes and ID 30 minutes respectively.

Figure 1 visualizes effects of treatments on extracted DNA concentration and A260/280 absorbency

Figure 1. Effects of treatments on DNA concentration and absorbency ratio (A260/A280); (A) Effect of 2-ME, CTAB, and incubation durations on DNA concentration; (B) Effect of 2-ME, PVP, and incubation durations on DNA concentration; (C) Effect of 2-ME, CTAB, and incubation durations on absorbency ratio of extracted DNA; (B) Effect of 2-ME, PVP, and incubation durations on absorbency ratio of extracted DNA

a

A

B

C

D

a Confidence interval

Figure 1 illustrates means of treatments for DNA quality and quantity. Based on Figures 1 A, B, C and D generally DNA quantity was more sensitive than quality to the treatments. In general 95% confidence interval (CI) for absorbency ratios were stable than DNA concentrations 95% CI.

After analysing first experiment, second experiment was designed to find optimum regents for the extraction buffer and to find right incubation duration in 65 áµ’C. The results obtained from the analysis of variances of second experiment are presented in Table 3. Analysis of variance for the second experiment results, revealed that although all the treatments was successful in extraction of high quality and high amount of DNA, there was no significant differences among treatments except for PVP and C-I. Increasing PVP concentration from 2% to 3 % had negative influence on amount of extracted DNA; extraction buffer with 2% PVP yielded 2901.6 ng/µl DNA whereas extraction buffer with 3% PVP yielded 2630.4 ng/µl (Table 4). The experiment revealed that one time application of C-I is enough for precipitation of DNA during extraction procedure. One time application of C-I yielded 3027.2 ng/µl DNA while two time application of C-I decreased DAN yield to 2504.8 ng/µl (Table 4).

Table 3. Analysis of variance results for experiment one.

Source of variations

Mean Square

(DNA concentration)

Mean Square

(260A/280A absorbency )

Replication

428244.9n.s

0.0040*

2-MEa

273506.3n.s

0.0010 n.s

C-Ib

3275186.8*

0.0006 n.s

2-ME*C-I

29210.3 n.s

0.0008 n.s

PVPc

882891.6**

0.0005 n.s

2-ME*PVP

2798.4 n.s

0.0014 n.s

PVP*C-I

31411.2 n.s

0.0014 n.s

2-ME*PVP*C-I

40130.5 n.s

0.0001 n.s

CTABd

1424.6 n.s

0.0010 n.s

2-ME*CTAB

191508.7 n.s

0.0012 n.s

CTAB*C-I

248184.4 n.s

0.0012 n.s

2-ME*CTAB*C-I

117602.1 n.s

0.0030n.s

PVP*CTAB

43941.1 n.s

0.0002 n.s

2-ME*PVP*CTAB

263.6 n.s

0.0001 n.s

PVP*CTAB*C-I

118415.3 n.s

0.0008 n.s

2-ME*PVP*CTAB*C-I

164865.2 n.s

0.0018 n.s

Coefficient of Variation

14.8 %

1.71 %

n.s. Non-significant

* Significant in 5%

** Significant in 1%

a 2-Mercaptoethanol

b chlorophorm-isoamyl alcohol

c Polyvinylpyrrolidone

d Hexadecyltrimethylammonium bromide

Table 4. Means of treatments for experiment 1.

DNA concentration ng/µl

260A/280A absorbency

Treatment

Mean

Std. Dev.

Mean

Std. Dev.

2-MEa3

2841.5

530.2

1.95

0.047

2-ME 6

2690.5

445.8

1.94

0.015

PVPb 2%

2901.6

474.1

1.95

0.045

PVP 3%

2630.4

478.1

1.94

0.018

CTABc2%

2771.4

543.1

1.94

0.019

CTAB 3%

2760.6

443.5

1.95

0.045

C-Id1

3027.2

356.7

1.94

0.019

C-I 2

2504.8

530.2

1.95

0.045

a 2-Mercaptoethanol

b Polyvinylpyrrolidone

c Hexadecyltrimethylammonium bromide

d Chlorophorm-isoamyl alcohol

Figure 2. Effects of treatments on DNA concentration and absorbency ratio (A260/A280); (A) Effect of 2-ME, CTAB, and incubation durations on DNA concentration; (B) Effect of 2-ME, PVP, and incubation durations on DNA concentration; (C) Effect of 2-ME, CTAB, and incubation durations on absorbency ratio of extracted DNA; (B) Effect of 2-ME, PVP, and incubation durations on absorbency ratio of extracted DNA

A

B

C

D

a Confidence interval

Table 4. Means of treatments for experiment 1.

DNA concentration ng/µl

260A/280A absorbency

Treatment

Mean

Std. Dev.

Mean

Std. Dev.

2-MEa3

2841.5

530.2

1.95

0.047

2-ME 6

2690.5

445.8

1.94

0.015

PVPb 2%

2901.6

474.1

1.95

0.045

PVP 3%

2630.4

478.1

1.94

0.018

CTABc2%

2771.4

543.1

1.94

0.019

CTAB 3%

2760.6

443.5

1.95

0.045

C-Id1

3027.2

356.7

1.94

0.019

C-I 2

2504.8

530.2

1.95

0.045

a 2-Mercaptoethanol

b Polyvinylpyrrolidone

c Hexadecyltrimethylammonium bromide

d chlorophorm-isoamyl alcohol

ME

PVP

CTAB

ng

A260/280

PVP

.781**

CTAB

.795**

.815**

ng

-.495**

-.656**

-.556**

A260/280

-.295**

-.311**

-.215*

.193

A230/260

-.364**

-.535**

-.450**

.633**

**. Correlation is significant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).