Insect Science (2010) 17, 465470, DOI 10.1111/j.1744-7917.2010.01318.

SHORT COMMUNICATION

A cost-effective, simple and high-throughput method for DNA extraction from insects
Malgorzata Lagisz, Gordon Port and Kirsten Wolff
School of Biology, Newcastle University, Newcastle upon Tyne, UK

Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 Ldeep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present. Key words DNA, extraction, genetic, isolation, molecular, technique

Introduction Molecular techniques are becoming increasingly important in insect research, including taxonomic, population genetics and evolutionary studies. Many such studies require large numbers of samples hundreds or even thousands and DNA extraction often becomes the limiting step in research in terms of time, money and equipment access. Traditional single-tube extraction methods are usually cheap and accessible, but they may be laborious and time-consuming or use hazardous chemicals (e.g., Thomsen et al., 2009). The single-tube extraction methods that are not laborious, time-consuming or hazardous, for example, Chelex resin extraction (Walsh et al., 1991), may not be suitable for long-term storage of isolated DNA samples or may leave polymerase chain reaction (PCR) inhibitors in the samples (Hoy, 2003). Commercial kits differ in their performance and cost-effectiveness (e.g., Ball &

Correspondence: Malgorzata Lagisz, School of Biology, Ridley Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK. Fax: +44 191 222 5229; email: losialagisz@yahoo.com

Armstrong, 2008). Most kits are prohibitively priced and/or involve use of specialist and expensive laboratory equipment. For example, recently proposed commercial and in-house high-throughput methods have utilized automatic liquid-handling robots (Ivanova et al., 2006), sonicators (Hunter et al., 2008) or mixer-mills (Hoarau et al., 2007; Whitlock et al., 2008), which are not available in most small laboratories. Additionally, 96-well plates capable of holding large volumes (>200 L) of liquids are recommended in most of the high-throughput methods. Such plates are at least several times more expensive than standard 200 L-deep 96-well plates that are routinely used for PCR. Theoretically, the use of large-volume 96-well plates may be avoided by reducing volumes of liquids used during extraction. Less liquid can be used when small amounts of tissue with low contents of secondary metabolites are processed, for example, from insects which are rarely large and do not contain many PCR inhibitors (Juen & Traugott, 2006). This indicates that there is an opportunity for developing a fast, simple and inexpensive extraction method that can yield high-quality DNA from insect tissues by combining traditional cheap single-tube and more recent high-throughput methods. To be useful for large-scale projects on tight budgets, such a method

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must use only inexpensive, non-toxic and easily accessible chemicals, consumables and very basic laboratory equipment. As a consequence, a method fulfilling these criteria will be also suitable for use in small laboratories with minimal resources, for example, in developing countries, field stations or for student projects. The goal of this paper is to present such a non-hazardous low-cost high-throughput and reliable DNA extraction procedure. The extraction process is based on a modified salting-out procedure (Miller et al., 1988) and an ammonium acetate precipitation method (Sambrook & Russel, 2001). The salt/alcohol precipitation used to isolate highquality DNA has a very low per-sample cost, and does not involve use of toxic chemicals. The only specialized equipment that is needed is a centrifuge with a rotor suitable for microtitre/PCR plates. We show that the method isolates DNA of substantial quantity and quality for use in PCR reactions. The added advantage is that the procedure is suitable for a range of insect species, for different life-stages and preservation methods. Materials and methods Insect material Fresh individuals of the red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), pea aphid Acyrthosiphum pisum (Harr.) (Homoptera: Aphididae) and diamondback moth Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) were collected from laboratory stock cultures maintained at the Newcastle University, UK. Specimens of Drosophila canadiana (Tahada and Yoon) (Diptera: Drosophilidae) were originally purchased from the Tucson Drosophila Stockcentre (Arizona, US) and had been stored in 99% ethanol at room temperature for approximately 3 years prior to the extraction. Before the start of DNA isolation, ethanol was evaporated for 15 min at room temperature from the fly samples. Live specimens of other insect species were killed by freezing at 18 C for approximately 15 min. Whole adults and larval stages of T. castaneum and adults and pupae of P xylostella were . used in the test extraction to test for differences in the method performance between the life stages and different amounts of tissue. Prior to DNA extractions, eight randomly selected individuals from each sample type (species, stage, wet/dry) were weighted to the nearest 0.01 mg, dried at 80 C for 24 h and weighted again to determine the dry tissue weight. Mean wet (for fresh samples) and dry tissue weights were calculated and used for evaluation of DNA extraction efficiency.

Fig. 1 Arrangement of samples in the 96-well extraction plate. White circles control wells (no specimens). Black circles wells with insect specimens (one per well). Plate row B Tribolium castaneum adults; row C Tribolium castaneum larvae; row D Acyrthosiphum pisum adults; row E Drosophila canadiana adults; row F Plutella xylosytella adults; row G Plutella xylosytella pupae.

Plate setup for extraction and cross-contamination test The major risk when using 200 L-deep 96-well PCR plates for DNA extraction comes from crosscontamination between neighboring wells during grinding, mixing and liquid handling. Therefore, our method was optimized for minimizing this risk and was tested for occurrence of contamination. A grid-like distribution of tissue samples on a plate was utilized, with blank/negative control wells (containing no tissue samples) interspersing and surrounding the wells containing insects (Fig. 1). One specimen per well of each species/life stage were placed in the rows BG, columns 211. DNA extraction protocol All samples (including controls) were homogenized in 10 L of DIGSOL extraction buffer (20 mmol/L edetic acid [EDTA], 50 mmol/L Tris (pH 7.5), 0.4 mol/L NaCl, 0.5% sodium dodecyl sulfate [SDS]) containing proteinase K (10 mg/mL, 20 : 1 mix ratio). The homogenization was performed manually using a multi-channel pipette fitted with 200 L tips with sealed ends. The ends of the pipette tips were sealed by moving the pipette with clean tips over a gas flame until a small ball of melted plastic formed at the end of each tip. A new set of tips was used for every column/row. After thorough homogenization, an additional 20 L of DIGSOL/proteinase K mix was added to all wells. The plate was sealed with adhesive PCR film (AB-0558, ABgene ThermoScientific, Epsom, UK), gently mixed on a vortex and left overnight to digest at 37 C in an oven (an incubator, heat block or thermocycler could be used as alternatives). The overnight digestion

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can be replaced by 3 h at 55 C. After incubation, 50 L of 4 mol/L ammonium acetate solution was added to all plate wells, the plate was sealed again and the contents mixed gently on a vortex. The plate was left at room temperature for 3060 min and then spun at maximum speed (1 520 g) for 15 min to pellet tissue debris in a centrifuge fitted with a 96-well plate rotor. The DNA-containing supernatant was transferred to a new plate using a multichannel pipette. The DNA was precipitated with 70 L of 99% ethanol. After sealing and gentle mixing on a vortex, the plate was centrifuged at maximum speed (1 520 g) for 15 min. The supernatant was removed from the plate by inverting the plate over a sink, and then tapping the plate gently on a stack of dry paper towels. The samples were then washed with 100 L of 70% ethanol. After that, the plate was dried for 5 min at 95 C on a heat-block. The pellet was resuspended in 100 L TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 7.58.0) and stored at 4 C. This DNA solution was used directly in the PCR reactions. For samples with very high DNA concentration a further dilution is recommended before PCR. In order to compare the performance of our protocol with traditional single-tube methods for the same type of tissue, we extracted DNA from T. castaneum adults using: (i) a commercial kit (DNeasy blood and tissue kit, QIAGEN, Crawley, UK); and (ii) a version of the ammonium acetate extraction protocol for T. castaneum with volumes of reagents suitable for 1.5-mL tubes (250 L of DIGSOL + proteinase K, 100 L of 4 mol/L ammonium acetate; 1 mL of 100% ethanol; 1 mL of 70% ethanol).

a 2% agarose gel containing ethidium bromide; a 100 bp DNA ladder (HyperLadder IV, Bioline, London, UK) was used as a size marker. Results DNA yield and purity The average total amount and quality of DNA extracted from each sample type (species/life stage) are shown in Table 1. All samples produced good yields of DNA minimum 0.5 g/insect and at least 0.5 g/1 mg of dry insect tissue, and up to maximum 5.0 g/insect or per mg of tissue. Such amounts are sufficient for multiple PCR reactions and are comparable with these obtained by a single-tube CTAB method optimized for DNA extraction from insects (Reineke et al., 1998). We observed some variation in yield among the individuals of the same species, which might reflect the between-well differences in levels of tissue disruption or in volumes of supernatant transferred between the stages of DNA extraction. The DNA quality measured as A260 /A280 ratio in spectrophotometric analysis was generally good, namely above 1.7 (Table 1), except for the extracts from P xylostella . adults and pupa. Suspected contamination with protein in the latter case might have resulted from the relatively large amount of tissue used for the extraction as well as from the high protein content in the pupa stage. Taking into account that the method used for the test extraction was specifically optimized for DNA isolation from T. castaneum adults, it is not surprising that morphologically and chemically different species and life stages give sub-optimal results. In the comparison to our protocol with traditional single-tube methods applied for T. castaneum adults, both the commercial kit and the ammonium acetate extraction gave higher yields of genomic DNA (average 5.4 1.9 g and 13.4 4.7 g, respectively, both

Amount and quality of DNA The quantity and the quality (A260 /A280 ratio for protein impurities) of the extracted DNA were measured with a spectrophotometer (BioPhotometer, Eppendorf, Hamburg, Germany). A 658-bp region of the COI gene was amplified by PCR using universal arthropod primers LCO1490 (forward) and HCO2198 (reverse) (Folmer et al., 1994). Amplifications were performed in a new 96-well PCR plate. Each 10 L PCR reaction mix contained 1 L of template DNA, 1 L of 10 Mg-free reaction buffer (Bioline, London, UK), 3 mmol/L MgCl2 , 2 mmol/L of each dNTP (Bioline, London, UK), 10 pmol of each primer and 0.25 U of Taq DNA polymerase (Bioline, London, UK). The PCR thermal program consisted of 94 C for 3 min, followed by 30 cycles of 94 C for 30 s, 50 C for 30 s, 72 C for 1 min, and 60 C for 30 min as a final extension after the last cycle. The amplified fragments were visualized by running 10 L of the PCR products on
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Table 1 Yield and purity (A260 /A280 ratio) of DNA extracted from four different insect species (mean SD, n = 5). Sample type Total DNA (g) A260 /A280 1.89 0.18 1.99 0.38 1.81 0.18 1.70 0.17 1.47 0.26 1.06 0.38

Tribolium castaneum adult 0.80 0.30 Tribolium castaneum larvae 9.61 6.77 Acyrthosiphum pisum adult 3.67 2.38 Drosophila canadiana adult 1.88 0.72 Plutella xylostella adult 6.93 11.31 Plutalla xylostella pupae 18.70 10.78

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n = 8) than the 96-well plate protocol (0.8 0.3 g, Table 1). Unexpectedly, for both single-tube protocols the quality of the DNA extract was lower (1.74 0.17 and 1.72 0.15 A260 /A280 ratios, respectively) than the values obtained for T. castaneum in the 96-well extraction (1.89 0.18, Table 1). PCR amplifications The isolated DNA was successfully amplified with universal mitochondrial COI primers for all insect samples tested. Figure 2 shows the PCR products for the whole extraction plate, including the negative control samples surrounding and interspacing the insect samples. The product sizes were as expected in all cases. The amplification was consistent, even for the species where DNA quantity was lowest and where absorbance ratio was below 1.7 A260 /A280 ratio, although there was some variation among samples that can possibly be attributed to gel loading errors. Tests for cross-contamination In the spectrophotometric analysis, wells used as negative control showed no readable quantity of DNA, indicating that no significant DNA cross-contamination occurred between the wells. At the same time, a PCR with COI mitochondrial primers gave an unexpected band in one of the negative controls (Fig. 2 position E10), suggesting that some DNA was present there. However, when a PCR reaction and a gel electrophoresis were repeated two more times for the whole row, no cross-contamination was observed. Therefore, the band on the first gel was probably due to the handling error during the setup of the first PCR or later during the gel loading, and not during the DNA extraction phase. Discussion According to our results, the proposed extraction method using ammonium acetate and ethanol in 96-well PCR plates proved to be simple and suitable for obtaining DNA of good quality which can be easily amplified by PCR. Our extractions were comparable in success and yield to these performed using singletube methods while bringing a range of benefits. The developed method employs inexpensive and non-hazardous reagents and is suitable for routine use with large numbers of samples. Furthermore, extraction and purification steps are accomplished with standard 96-well PCR plates

with possible scope for automation of the procedure. This also greatly facilitates both storage and downstream processing (e.g., PCR setup). The method is applicable to different types of insect tissues. Grinding of samples and liquid transfers using a multi-channel pipette allow for significant reduction of time necessary for extraction, relative to the traditional version of the single-tube ammonium-acetate method. The total extraction time (extraction and purification, excluding overnight incubation) for 2 96 samples is approximately 4 h, although more sets of plates can be processed on the same day resulting in only slightly longer total extraction time. Material costs (including plastic ware) are less than USD $0.25 per sample. Cross-contamination is avoided by using small volumes of reagents and very careful handling of the plates. When developing the protocol, we tried different types of plate seals and found that the stickiness of the adhesive plate seal is very important too weak adhesion allows the liquids to evaporate during incubation or to leave the well when mixing. Too strong adhesion makes it difficult to remove the sealing film and increases the risk of creating a spray of droplets. The adhesive PCR film AB0558 produced by ABgene (ThermoScientific, Epsom, UK) worked best for us and therefore was used in all subsequent DNA extractions. The method described was developed as a first step of a larger population genetics project on the red flour beetle, T. castaneum. For the DNA extractions we used individuals that were either freshly killed or stored for some time after air-drying. Body parts of T. castaneum adults (head, abdomen) were also used. DNA from several thousands of individuals was extracted by one person within several weeks. We successfully amplified the extracted DNA in PCR reactions with a range of nuclear primers: microsatellite and sexing markers, also in multiplex reactions. Some PCRs were run using template DNA that was kept in a fridge at 4 C or in a freezer at 18 C for a few months. In all cases, satisfactory results were obtained. The most important feature of the proposed method lies in the fact that it does not require specialist equipment other than a centrifuge capable of spinning plates. While many other high-throughput extraction methods published so far can offer similar or better efficiency, they usually rely on some type of non-standard equipment, like sonicators, mills or robots. In other cases expensive deep-well plates are required. The cost of purchasing such non-standard items may be particularly prohibitive for researchers from developing countries, small laboratories and field stations.

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Fig. 2 Agarose gel (2%) with extracted DNA from four insect species amplified with mitochondrial COI primers. Gel lanes contain 10 L PCR reactions or 5 L of 100-bp HyperLadder IV (Bioline, London, UK). Arrangement of the samples is the same as on the 96-well PCR plate (Fig. 1) letters represent row labels from the extraction plate, while numbers represent column labels.

Acknowledgments This work was funded via an EU Marie Curie IntraEuropean Fellowship (MEIF-CT-2005-024701).

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