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Draft for submission to Molecular Ecology Resources
The need for 2-mercaptoethanol in DNA extraction of molluscs
Danabalan R, von Rintelen T.
Museum für Naturkunde Invalidenstraße 43 10115 Berlin, Germany
Keywords: 2-Mercaptoethanol, DNA extraction, mucopolysacchrides
Renita Danabalan
Museum für Naturkunde
Invalidenstrasse 43
10115 Berlin, Germany
Tel: + 49 30 ´2093 8430
email: [email protected]
Use of βME in CTAB
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Introduction
The main challenge in the extraction of genomic DNA from any organism is the removal of
proteins and oxidating phenolic compounds (Katterman and Shattuck, 1983). In the case of
molluscs, the co-purification of mucopolysaccharides with genomic DNA (Popa et al., 2007;
Pereira et al., 2011) presents an added challenge. Polysaccharides a sugar compound present
in molluscs in varying degrees, has been shown to inhibit enzymatic reactions such as
polymerases (Sokolov, 2000).
At present the most common method of purifying genomic DNA from molluscs is based on
the protocol modified by Winnepenninckx et al (1993) using a lysis buffer containing
hexadecyltrimethylammonium bromide (CTAB), 2-mercaptoethanol (βME) as well as a high
salt concentration (1.4M Nacl). The use of βME in the CTAB buffer (Williams et al., 2003;
Williams &Reid., 2004; Goodacre., 2012) in molluscs is integral to the extraction process
(REFS); It is a key chemical used in many extraction buffers to inhibit oxidation processes
and denature proteins during lysis (Sa et al., 2011). However, the availability of Proteinase K,
performing a similar function as βME questions the need of this toxic chemical in
laboratories.
More recently, several extraction techniques such as including DNAzol (Kirkendale., 2009),
Qiagen Blood and tissue (Christian et al., 2007; Doherty et al., 2007), Puregene kit (Henley et
al., 2006; McCartney et al., 2009) are being used on molluscs. This, together with the
advancement of novel automated systems present in the industry requires the reassessment of
gold standards. One of these methods is a modified solid phase reversible immobilisation
(SPRI) technique, using carboxyl coated magnetic beads to preferentially bind to nucleic
acids post lysis (Uhlen, 1989; Hawkins et al., 1994; Biosprint, Qiagen); thus eliminating the
need for chemicals such as βME, phenol and chloroform and bringing us one step closer
towards the use of automated extraction techniques.
As such, the primary aim of this study was to determine the need for βME in the DNA
extraction of fresh molluscs from 3 genera by measuring the concentration of DNA and the
amplification and sequencing of a 660bp mitochondrial Cytochrome Oxidase I (barcoding)
region. Second to that was to determine the feasibility of using an automated extraction
system on mollusc tissue; comparing the concentration of DNA to that of the CTAB extracted
tissue
Methods and Materials
Specimens used in this study comprised of freshly collected Viviparus contectus, Cepaea
nemoralis and Dreissina polymorpha. from Brandenburg, Germany preserved in both 100%
laboratory grade ethanol and denatured ethanol.
A 1mg piece of foot and mantle tissue was taken from one sample per species from both
preservation methods and treated accordingly: Overnight lysis in CTAB buffer with 0.02%
βME (n=12) followed by phenol-chloroform extraction according to Winnepenninckx et al.
1993, overnight lysis in CTAB lysis buffer without 0.02% βME (n=12) followed by phenolchloroform extraction and overnight lysis in CTAB buffer without 0.02% βME followed by a
solid phase reversible immobilisation (SPRI) based extraction (Biosprint, Qiagen, Germany;
n=24, each sample was replicated) according to the manufacturer’s instructions.
Genomic DNA was analysed using a DNA fragment analyser (Advanced Analytical,
Oaklahoma) and concentration (ng/µl) of the fragments of DNA present in every sample was
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measured; for replicate samples extracted using the Biosprint, DNA concentration from the
respective specimen was pooled and the mean DNA concentration was used for subsequent
analysis. The concentration of DNA from samples extracted using CTAB with βME and
without βME were compared using paired t-test (SPSS v. 170.) and DNA concentration from
βME, samples without βME and those extracted from the Biosprint were compared using
Wilcoxon Signed Ranks test (SPSS v. 17.0).
Amplification of the 660bp Cytochrome Oxidase I barcoding region was also carried out from
all specimens using LCO1490 (Folmer et al., 1994) and HCO2198 var (Rintelen et al., 2004)
in final PCR volume of 20ul containing 1X Buffer*, 0.2mM dNTPS, 0.4µM primer each,
1mM MgCl2 and 1 units of Taq*. Successful amplifications were then cleaned using 2µl of a
1:5 dilution of exosap IT* (*New England Biolabs) with 8µl of PCR product, the mixture was
then incubated at 37°C for 15 minutes, after which the enzymes were inactivated at 80°C for
20mins. From this 2µl o cleaned product was then sequenced (ABI 3310, Applied
Biosystems) in both directions to ensure the correct amplification of target fragment.
Sequences were analysed using Codeoncode Aligner v3.7.1 (LI-COR inc).
Results
Concentration of extracted DNA
In general the concentration of DNA varied the most in samples that were extracted using the
Qiagen, Biosprint method; values ranged from 0.377ng/µl to 41.97ng/µl (Table 1). Samples
extracted using the CTAB buffer with and without βME had more consistent concentrations
averaging at 0.0710ng/µl for samples extracted with βME and 0.0736ng/µl for samples
extracted without βME.
Statistical analysis
The mean DNA concentration was compared using paired t-tests on samples extracted with
and without Beta-mercaptoethanol (t0.05(2), 11= 0.0593) and samples preserved in denatured
alcohol and absolute ethanol: βMEdenatured vs n βMEETOH- t0.05(2),5= 0.490, p=0.645;
βMEdenatured vs nβMEdenatured- t0.05(2),5=0.094, p=0.928: βMEETOH vs nβMEETOH- t0.05(2), 5= 0.746; p= 0.489; βMEdenatured va nβMEETOH- t0.05(2),5= -0.419; p=0.692. No significant
difference found between denatured and absolute ethanol or samples extracted with and
without βME.
Wilcoxon’s signed rank test also showed no significant difference between the extraction of
CTAB with or without βME. However, there was a significant difference between
concentration of DNA between samples extracted using the Qiagen Biosprint and CTAB with
βME (Z0.05(2),12= -2.589, p=0.010) and without βME (Z0.05(2),12= - 2.353, p=0.019).
Amplification of mitochondrial Cytochrome Oxidase I
Cytochrome Oxidase I was amplified from 54 out of the 72 samples extracted; 83% of the
samples that amplified were extracted without βME (n=11 CTAb; n=36 Biosprint; Table 2).
Discussion
Beta-mercaptoethanol has been a component in the extraction of mucopolysacchride rich
tissue. This is the first study to date ascertaining the need for the reagent in the lysis of
molluscan tissue and the impact of extraction done without the use of ΒME measured in
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paired samples. The results from this study indicate that specimens could be extracted using
with and without βME without subsequent impact on PCR amplification and sequencing.
It was observed that higher concentrations of DNA were obtained from samples extracted
using the SPRI method (Biopsrint, Qiagen). This was also reported by Pereira et al., 2011;
they attributed this to a reduction human involvement in automated systems compared to the
phenol-chloroform extraction method. While samples extracted using the CTAB buffer,
followed by a phenol-chloroform extraction showed little variability in concentration of DNA,
sequences obtained were variable even within extracted foot and mantle samples of the same
specimen Interestingly, concentrations of DNA between samples preserved in denatured
ethanol and 100% ethanol showed no significant difference, even though the majority of
samples that resulted in good sequences arose from those preserved in 100% ethanol.
Studies conducted on extraction methods of molluscs using commercial kits and precipitation
(Popa et al., 2007; Sokolov, 2000, Geist et al., 2008)) without the use of 2-mercaptoethanol,
have determined the quality of DNA by measuring the A260/280 ratio as well as PCR
amplification. In general, as also noted by Popa et al. (2007), the absorbance value did not
have any impact on amplification.
For the most part, DNA extracted from specimens collected in this study, amplified the full
660bp COI barcoding fragment. However, obtaining sequences for the amplified products
proved to be challenging even for samples extracted with ΒME. This could be attributed to
the loss of DNA in the aspiration of the supernatant phase using the phenol chloroform
method, however sample size per specimen is too small to come to any definite conclusion.
What can be said with certainty is the use of βME in the lysis buffer for molluscs is not
essential; as mentioned earlier, the CTAB extraction method was adapted from a plant
protocol, in a plant extraction βME is essential in preventing the degredation of DNA by
polyphenolic compounds present in the leaves (Dehestani and Tabar, 2007; Sa et al., 2011).
These chemicals are largely found in the digestive gland of molluscs which are not commonly
used in molecular genetics as are foot and mantle tissue. Taking this into consideration the use
of 2-Mercaptoethanol can be omitted from the extraction process and automated techniques
such as the Biosprint Plant kit (Qiagen) used in this study could be used in high-throughput
extraction of molluscs.
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Table 1: DNA concentration (ng/µl) of samples extracted using CTAB lysis with βME and
without βME followed by a phenol chloroform and SPRI based extraction
Concentration of DNA (ng/µl)
CTAB with βME
CTAB nβME
Biosprint
samples
denatured 100% denatured 100%
denatured
100%
C. nemoralis (foot)
0.1006
0.0623
0.0736
0.0879
0.377015
0.083
C. nemoralis (mantle)
0.0547
0.0648
0.0600
0.0558
0.36898333 1.17548667
V. contectus (foot)
0.0508
0.0576
0.0484
0.0650
0.04746667
0.075425
V. contectus (mantle)
0.0684
0.0665
0.0776
0.0770
0.08754
41.974475
D. polymorpha (foot)
0.0867
0.0840
0.0852
0.0610
0.63234524
0.079475
D. polymporpha
(mantle)
0.0756
0.0803
0.0887
0.1030
0.20446429
0.064525
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Table 2 Number of mitchondiral COI (660bp) amplified and sequenced (in paranthesis) for
extraction using CTAB buffer with and with βME and Biosprint (SPRI).
samples
C. nemoralis
(denatured
ETOH)
C. nemoralis
(ETOH)
V. contectus
(denatured
ETOH)
V. contectus
(ETOH)
D. polymorpha
(denatured
ETOH)
D. polymorpha
(ETOH)
CTAB
With βME n=12
Without βME n=12
2 (2)
2 (1)
Biosprint n=48
Total
7(2)
11(5)
2(1)
2(2)
7(5)
11(8)
1
2
4
7
2(1)
2
4(4)
8(5)
2(2)
2(2)
8(7)
12(11)
2
2(2)
8(7)
12(9)
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