185,147-150

ANALYTICALBIOCHEMISTRY

(1990)

Isolation of High-Molecular-Weight

DNA from Insects

Jacquelyn

L. Ridgway

M. Henry,l

Insect Chemical

Received

August

Ashok

K. Raina,

and Richard

Ecology Laboratory,

Agricultural

Research Service, USDA, Beltsville,

Maryland

20705

2,1989

A simple and rapid method for the isolation of highmolecular-weight DNA from insects is described. The method does not require CsCl ultracentrifugation or extensive dialysis. High-molecular-weight DNA was obtained within 24 h. Since the entire insect was used for DNA isolation, an initial nuclei-enriched fraction was required. Genomic DNA was extracted from lysed nuclei by organic phase separation (liquid/liquid extraction). This method has been successfully applied to the isolation and purification of DNA from eight different adult insects (Heliothis zea, Musca autumnalis, M.

domestica, Blatta orientalis, Tenebrio molitor, Lymantria dispar, Ostrinia nubilalis, and Manduca se&a). The recovered DNA can be cleaved with restriction endonucleases, ligated efficiently using standard cloning vectors, and hybridized to synthetic oligonucleotides. 0 1990 Academic Press, Inc.

method has the advantages that it avoids CsCl ultracentrifugation or extensive dialysis and it is economical and reproducible. High-molecular-weight DNA has been effectively isolated and purified from H. zea adults and seven other adult insect species [face fly, Musca autumn&; oriental cockroach, Bluttu orientalis; yellow mealworm, Tenebrio molitor; housefly, M. domesticu; gypsy moth, Lymuntriu dispur; European corn borer, Ostriniu nubilulis; and tobacco hornworm, Munducu sextu]. DNA isolated by this method has been successfully used in the construction of a genomic library, restriction analysis for Southern blotting (8), and hybridization to a synthetic 33-bp mixed oligonucleotide probe developed from the pheromone biosynthesis activating neuropeptide (PBAN)3 amino acid sequence (9). MATERIALS

AND

METHODS

Enzymes and Chemicals The isolation of high-molecular-weight DNA is essential for the construction of genomic libraries and Southern blotting. At present there are a limited number of protocols for the isolation of high-molecular-weight DNA from insects (1,2). Protocols for the isolation of eukaryotic genomic DNA are often time-consuming, expensive, and designed for cultured cells and tissues (37). Standard procedures for extraction and purification of intact high-molecular-weight DNA from eukaryotic organisms require enzyme digestion or detergent lysis, multiple organic solvent extractions, CsCl ultracentrifugation, and extensive dialysis. Several attempts to isolate DNA from the corn earworm moth, HeZiothis zeu, using above methods proved of limited success. Subsequently, a protocol for the isolation of genomic DNA from insects that does not rely on labor-intensive methodology and expensive equipment was developed. The 1 Present address: Physiology Medical Center, Washington, ’ To whom correspondence

Branch, USAIDR, DC 20307. should be addressed.

0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

Inc. reserved.

Walter

Proteinase K, RNase Tl, restriction endonucleases, and bacteriophage X DNA were purchased from Bethesda Research Laboratories, and pancreatic RNase A from Boehringer-Mannheim. Oligonucleotides were synthesized using a Biosearch 8750 DNA synthesizer. [T-~~P]ATP (3000 Ci/mmol) was obtained from New England Nuclear. T4-infected Escherichiu coli polynucleotide kinase was purchased from Pharmacia. Preparation

of Nuclei

Two grams of frozen (-80°C) dewinged insects were ground to a powder with a mortar and pestle embedded in dry ice. All subsequent procedures prior to lysis were performed at 4°C. The frozen material was thoroughly homogenized in 30 ml of TENT buffer (10 mM Tris-Cl, pH 7.4; 25 mM EDTA; 10 InM NaCl; 0.5% Triton X-100) using a Ten Broeck homogenizer. Debris was removed by filtering the homogenate through 10 layers of sterile

Reed Army 3 Abbreviations used: PBAN, pheromone neuropeptide; SDS, sodium dodecyl sulfate.

biosynthesis

activating

147

148

HENRY, Nuclei-enriched

RAINA,

fraction

PCIA

--incubate

37OC

aqueous

Isolation

phase

t extract --remove

aqueous

phase

t Precipitate

DNA --spool --dissolve

Store -------------r Agarose Southern

I

blot

I at

in

TE buffer

4OC ---e---mmm-__

1

gel

in mM

K

extractC--------------------------------remove

CIA

RIDGWAY

before. The pellet enriched in nuclei was resuspended pH 7.4; 25 10 ml of TEN buffer (10 mM Tris-Cl, EDTA; 10 mM NaCl) containing 1 mg/ml proteinase and used as a source of DNA.

--lyse I

AND

1 Remove

analysis

residual

RNA

--RNase A/RNase t Extract------------------.

FIG. 1. Outline of procedure of high-molecular-weight DNA enriched fraction.

Tl

used in the isolation and purification from insects starting with a nuclei-

cheesecloth (prewet with TENT buffer) and centrifuged for 5 min at 5000g in a swinging bucket rotor. The pellet was gently resuspended twice in 10 ml of TENT buffer with the use of a wide mouth pipet and centrifuged as

b

c

d

e

of DNA

SDS was added to the suspension of the nuclei to give a final concentration of 1%. The effectiveness of the nuclei-isolation procedure could be assessed by an increase in viscosity during the extraction of the DNA. The lysate was incubated at 37°C for 4 h with periodic swirling. Following incubation, 1 ml of 5 M NaCl was mixed with the lysate and an equal volume of phenol: chloroform:isoamyl alcohol (25:24:1, v/v/v) was added. The extract was gently inverted for 15 min and centrifuged to facilitate phase separation. The aqueous (upper) phase was transferred to a fresh tube using an inverted pipet and reextracted with an equal volume of chloroforrmisoamyl alcohol (2411, v/v) followed by 2.5 vol of cold absolute ethanol. The DNA was spooled onto a glass rod, rinsed twice with 5 ml of 70% cold ethanol, and finally dissolved in 10 ml of TE buffer (10 mM Trisat 4°C overCl, pH 8.0; 1 mM EDTA, pH 8.0) stationary night. Removal of Residual

RNA

Residual RNA was removed by incubating DNase-free RNase A (50 pg/ml) plus RNase Tl(l2.5 U/ml) with the

B

A a

and Purification

f

g

h

i j

48.5 + 23.1* 9.4-6.6+ 4.4-9

2.3-, 2.0+

FIG. 2. Agarose gel analysis of adult insect DNA prepared as described under Materials and Methods. (A) Undigested and (B) digested DNAs extracted from M. autumnalis, B. orientalis, T. molitor, M. domestica, L. dispar, 0. nubilalis, M. sexta, and H. sea (lanes c-j, respectively) were subjected to electrophoresis in a 0.8% agarose gel buffered with 0.89 M Tris-borate, pH 8.0, and stained with ethidium bromide (1 pg/ml). Digested (lane a) and undigested (lane b) bacteriophage X DNAs were used as molecular weight markers. Insect and bacteriophage DNAs were digested to completion with the restriction endonuclease HindI (10 U/pg). Sizes at the side are in kilobases.

RAPID

a

b

ISOLATION

OF

INSECT

ab

c

149

DNA

c

d

e

f

g

h

23.19.4+ 6.64.k

2.3* 2.0+

*:

FIG. 4. Autoradiograph obtained after dot blot analysis of adult insect DNA. Undigested DNA from each insect was serial diluted and hybridized with the 32P-labeled PBAN mixed oligonucleotide probe. The sources of the various DNA samples were M. autumnalis (a), B. orientalis (b), T. molitor (c), M. domestica (d), L. dispar (e), 0. nubilah (f), M. sextu (g), and H. zea (h). DNA concentration markers are in micrograms. FIG. 3. Restriction analysis of recombinant EMBLB DNA. An aliquot (10 ~1) of recombinant phage DNA isolated by the plate lysate method (5) was incubated either with (lane b) or without BamHl (lane c). DNA was subjected to electrophoresis in a 0.8% agarose gel and stained with ethidium bromide (1 pg/ml). Lane a represents molecular weight markers (bacteriophage X cut with HindIII). Sizes at the side are in kilobases.

DNA preparation at 37°C for 3 h. DNA was recovered in ethanol as described above and dissolved in TE buffer at 1 pg/pl. DNA concentration was determined by uv absorbance at 260 nm (10). DNA Blot Hybridization Digested DNA (lo-20 pg) was subjected to electrophoresis and transferred to nitrocellulose filters (5). Oligonucleotide probes were end-labeled to a specific activity of 6 X 10’ cpm/pg and used at a concentration of 1 X lo6 cpm/ml. Prehybridization and hybridization were carried out at 55°C in 6X SSC, 0.5% SDS, 1X Denhardt’s solution, and salmon sperm DNA at 100 pg/ml. Filters were washed 4X 15 min at 25°C and 60 min at 55°C in 6X SSC. Serial dilutions of undigested DNA (5 pg) were spotted onto nitrocellulose (saturated in 6X SSC) with the aid of a Minifold device (Schleicher & Schuell) for dot blot analysis. The filter was baked in a vacuum oven at 80°C for 2 h and hybridized as above. RESULTS

AND

DISCUSSION

High-molecular-weight DNA was isolated from H. zea and seven other species of insects starting with a nuclei-

enriched fraction as shown in Fig. 1. The initial preparation of nuclei lowered the amount of RNA present in the DNA preparation, hence, allowing Southern blot analysis to be performed without removal of residual RNA. DNA from various insects was subjected to agarose gel electrophoresis to determine if it was of high-molecular8 b c d e f g h i j

k I m

23.19.4 + 6.6+ 4.4+

FIG. 6. Autoradiograph obtained after Southern blot transfer of adult insect DNA. High-molecular-weight DNA from H. zea (lanes bg) and M. sezta (lanes h-m) was digested to completion with BamHl (lanes b and h), EcoRl (lanes c and i), Hi&II (lanes d and j), PstI (lanes e and k), HpaII (lanes f and l), and MspI (lanes g and m) and hybridized with the 32P-labeled PBAN mixed oligonucleotide probe. Bacteriophage h DNA (lane a) digested with Hind111 and end-labeled with [o(-32P]dCTP was used as a molecular weight marker. Sizes at the side are in kilobases.

150

HENRY,

RAINA

weight and of suitable purity for restriction analysis. As shown in Fig. 2A, the undigested DNA molecules migrated as one high-molecular-weight band similar to the 4%kb marker. Whereas, the digested insect DNA fragments were present throughout the entire length of the gel for each species examined (Fig. 2B). These results demonstrated the following: (a) isolated DNA was of high-molecular-weight, (b) DNA was suitable for endonuclease restriction analysis, (c) degree of mechanical shearing was minimal, and (d) the presence of small amounts of RNA did not inhibit the restriction endonuclease. To test the suitability of this high-molecular-weight DNA for cloning, an aliquot of H. zea RNase-treated DNA was partially digested with the restriction endonuclease Mb01 and ligated in EMBL3 vector (data not shown). The resultant genomic library had a packaging efficiency of 1 X 10” PFU/pg vector DNA which makes this DNA of acceptable purity and size for molecular applications involving restriction and cloning. Figure 3 shows the results of the restriction analysis with BamHl of the recombinant EMBL3 DNA. The digested recombinant phage DNA contained multiple bands ranging in size from 10 to 20 kb, whereas, the undigested recombinant phage preparation contained only one band. These results indicated that the genomic DNA inserts were successfully ligated into EMBL3 thus providing further evidence for the purity and integrity of the isolated highmolecular-weight DNA. To further substantiate the quality of the isolated DNA for molecular biology applications, serial dilutions of undigested DNA were hybridized with a synthetic 33bp mixed oligonucleotide probe to PBAN. As shown in Fig. 4, specific binding was observed for all eight insect species and the probe hybridized to DNA at a concentration as low as 0.3 pg. These results indicate that the isolated DNA was of suitable purity for DNA-DNA interactions. Since the PBAN probe significantly hybridized to undigested DNA, Southern blot analysis was performed using the same hybridization conditions. The probe did anneal to the digested DNA resulting in multiple bands for each insect species (Fig. 5). The results clearly indicate that this DNA could be denatured and renatured with foreign DNA. Multiple bands may indicate one or all of the following: (a) population polymorphism (since insects were pooled for DNA isolation), (b) presence of intervening sequences, (c) multiple gene copies, and (d) sequences other than PBAN. The genomic library is presently being screened in order to determine the nucleotide sequence of PBAN.

\ND

RIDGWAY

DNA isolated with the present method dissolved readily in TE buffer and was not pigmented. The 2601 280 nm ratio generally ranged between 1.7 and 1.8, indicating a relatively low level of contamination either by proteins or from reagents used in the extraction procedure. From 2 g of starting materia1 l-2 mg of high-molecular-weight DNA was obtained. The DNA may be stored indefinitely at 4°C. The procedure has been scaled down and applied successfully to other developmental stages of insects (data not shown). The conventional cesium chloride density gradient centrifugation methods are tedious, expensive, and time-consuming, requiring up to several days of work. The present method eliminated these steps and yielded high-molecular-weight DNA within 24 h. The procedure which involved detergent lysis and organic extractions of the nuclei-enriched fraction enabled us to obtain a higher yield of undegraded DNA when compared to other methods in which the homogenates were lysed in the presence of particulate matter and subsequently extracted with phenol and chloroform (1, 3-6). The primary advantages to this method are its reliability, reproducibility, and simplicity. From the results it is apparent that the DNA obtained was a good substrate for restriction enzymes and other DNA-modifying enzymes. ACKNOWLEDGMENT We thank Dr. Autar Mattoo tory for laboratory facilities.

of the Plant

Molecular

Biology

Labora-

REFERENCES 1. Vlasak,

R., Ullman,

C. U., Krieil,

G., and Frischauf,

A. M. (1983)

Eur. J. Biochem. 135,123-126. N., 2. Weeks, D. P., Beerman, chem.162,376-385. 3. Bowtell, D. D. L. (1987) Ad. 4. Johnson, P. H., Olson, C. B., them. Physiol. 813,1045-1051. 5. Maniatis, T., Fritsch, E. F.,

6.

and Griffith,

0. M. (1986)

Ad.

Bio-

Biochem. 162,463-465. and Goodman,

M. (1985)

Comp. Bb

and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 280-281, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Strass, W. M. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seldman, J. G., and Struhl, K., Eds.), pp. 221-222, Wiley, New York.

7. Kupiec, J. J., Giron, M. L., Vile&, D., Jeltsch, R. E. (1987) Anal. Biochem. 164,53-59.

J. M., and Ravier,

8. Southern, E. M. (1975) J. Mol. Bill. 98,503-517. 9. Raina, A. K., Jaffe, H., Kempe, T. G., Keim, P., Blather, R. W., Fales, H. M., Riley, C. T., Klun, J. A., R&way, R. L., and Hayes, D. K. (1989) Science 244,796-798. M. D., and Battery, J. F. (1986) Basic Meth10. Davis, L. G., Dibner, ods in Molecular Biologv. D. 327. Elsevier. New York.