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DNA ISOLATIONUSING METHIDIUM-SPERMINE-SEPHAROSE
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5. Add 30 ~g yeast tRNA per milliliter of D N A solution. 24 Add 100/zg of proteinase K and incubate at 50 ° for I to 2 hr. Extract twice with equal volumes of a 1 : 1 mixture of phenol-chloroform,18 and once with an equal volume of chloroform alone. Separate the layers by centrifugation at full speed in a microfuge at room temperature, saving the aqueous layer at each stage for the subsequent step. 6. Following these organic extractions, transfer the aqueous layer to a 1.5-ml microfuge tube. Add two volumes of ethanol and mix well. Store at - 70 ° for 30 min. Spin in a microfuge at 4 ° for 15 min to pellet down the D N A precipitate. 7. Resuspend the pellet in 400 /zl of T E containing 0.3 M sodium acetate (pH 7.0), and precipitate the D N A again, by mixing with 2 vol of ethanol. Store at - 7 0 ° for 30 min. Spin in a microfuge at 4 ° for 15 min to pellet down the D N A precipitate. 8. Dissolve the pellet in 40/xl TE. The solution contains low molecular weight D N A along with some RNA. The D N A preparation is suitable for diagnostic restriction digestions followed by analysis involving Southern blot hybridization using a labeled probe specific for the DNA. 25 If the isolated low molecular weight D N A is a bacterial plasmid, this D N A preparation is also suitable for reintroduction into bacteria by transformation. 12 Acknowledgment We thank Julie Yamaguchi and Daniel C. Alter for help with standardization of the boiling method and development of the phenol-chloroform lysis-extraction method, respectively, and Dr. Cho-Yau Yeung for helpful discussions. We thank Linda Cardenas for clerical assistance. This work was supported by grants from the National Institutes of Health, the American Cancer Society, and the University of Illinois at Chicago. 24The tRNA is added as a carrier at this stage to minimize loss of DNA during the organic extractions and to help in the precipitation of the DNA at later steps. 25E. M. Southern, J. Mol. Biol. 98, 503 (1975).
[4] D N A I s o l a t i o n U s i n g M e t h i d i u m - S p e r m i n e - S e p h a r o s e B y JOHN D. HARDING, ROBERT L. BEBEE, and GULILAT GEBEYEHU
Introduction Rapid, quantitative isolation of D N A from complex biological samples is required for many protocols in molecular biology and molecular diagnosMETHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[4]
tics. Standard methods using organic solvents such as phenol are reliable, but are time consuming and involve the use of toxic chemicals. Therefore many alternative methods for DNA isolation have been examined over the past several years. In designing novel reagents for rapid isolation of DNA from complex samples, we reasoned that a very efficient "capture reagent" would consist of an intercalating dye attached to a solid support by a molecular linker.l'2 The intercalator binds the DNA with high affinity and the solid support allows rapid separation of the bound material from unwanted contaminants. In this chapter we describe protocols for isolating DNA from a variety of complex samples using a DNA capture reagent consisting of the intercalator, methidium, attached to a Sepharose bead by a spermine linker. DNA is released from the reagent in 0.1 to 0.5 N KOH or NaOH and is characterized by procedures such as dot-blot hybridization, sequencing, or polymerase chain reaction analysis. Materials DNA capture reagent (methidium-spermine-Sepharose) and DNA extraction buffer [Cat. No. 80885A; Bethesda Research Laboratories (Life Technologies, Inc., Gaithersburg, MD)]: The DNA capture reagent is synthesized as described in Harding et al.l Proteinase K solution: 50 mg/ml proteinase K (Bethesda Research Laboratories) in 0.2 M Tris-HCl, 0.1 M NazEDTA, 3% (v/v) Brij 35; pH 7.5 TE buffer: 10 mM Tris-HC1, 1 mM Na2EDTA; pH 7.5
Methods Protocol 1: Isolation o f D N A from Serum or Urine and Characterization by Dot-Blot Analysis 1. To 50/xl of serum or urine in a 1.5-ml microcentrifuge tube add 40/zl of DNA extraction buffer and 10/xl of proteinase K solution. Incubate for 60 min at 65 °. 2. Vortex the stock tube of DNA capture reagent vigorously for a few seconds to suspend the capture reagent uniformly and pipette 50/xl of the slurry into the sample tube. Vortex the sample tube vigorously for a few i j. Harding, G. Gebeyehu, R. Bebee, D. Simms, and L. Klevan, Nucleic Acids Res. 17, 6947 (1989). 2 G. Gebeyehu, L. Klevan, and J. Harding, U.S. Patent 4,921,805 (1990).
[4]
DNA ISOLATIONUSINGMETHIDIUM--SPERMINE-SEPHAROSE
31
seconds and immediately place it on a rotator (such as a Labquake rotator, Labindustries Inc., Berkeley, CA) for 30 min at room temperature. 3. Spin the tube for 30 sec in a microcentrifuge (12,000 g) to pellet the capture reagent-DNA complex. Carefully remove the supernatant with a micropipette and discard it appropriately (serum and urine may be biohazards). 4. Add 100/~1 of TE buffer, vortex vigorously, and spin in a microcentrifuge for 30 sec. Carefully remove the supernatant with a micropipette and discard appropriately. 5. To the capture reagent pellet add 100 ~1 of 0.5 N NaOH. Vortex vigorously for a few seconds and place on a rotator for 10 min at room temperature. 6. Spin out the capture reagent in a microcentrifuge for 30 sec and carefully remove the supernatant, which contains the DNA, with a micropipette. 7. For alkaline dot blotting, presoak the membrane (nitrocellulose or nylon) in 0.5 N NaOH for 5 min with gentle agitation. Place the membrane on a dot-blot apparatus. Pass the sample, eluted directly from the capture reagent, through the membrane. Wash each dot-blotted sample with 500/~1 of 0.5 N NaOH. For nitrocellulose, bake the membrane for 1 hr at 80° in a vacuum oven. For nylon, treat the membrane according to the instructions of the manufacturer. Prehybridize and hybridize the membrane by standard protocols or using conditions previously optimized for a particular probe. 8. An alternative dot-blotting technique that has worked equally well is as follows. To the sample eluted from the capture reagent, add an equal volume of 2 M ammonium acetate. Ammonium acetate is prepared by dissolving the solid salt to a 2 M final concentration; the pH is not adjusted. Before applying the sample, soak the membrane briefly in distilled water and then for 5 min in 1 M ammonium acetate with gentle agitation. Place the membrane on a dot-blot apparatus. Pass the DNA sample through the membrane; wash each dot blotted sample with 500/xl of I M ammonium acetate. If the membrane is to be baked prior to hybridization (e.g., nitrocellulose and some nylons), incubate it in 20 × SSC (3 M NaCI, 0.3 sodium citrate, pH 7.0) with gentle agitation for 5 min at room temperature prior to the baking step. Protocol 2: Isolation of DNA from Whole Blood or Cultured Cells and Analysis by Polymerase Chain Reaction 1. Dilute 1 to 10/~1 of whole blood, collected in ethylenediaminetetraacetic acid (EDTA) as an anticoagulant, to a total volume of 50/xl with TE buffer in a 1.5-ml microcentrifuge tube. If cultured cells are to be
32
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[4]
analyzed, pellet the cells from the culture medium and resuspend the pellet in 50/xl of TE buffer. To either sample, add 40/xl of DNA extraction buffer and 10/~1 of proteinase K solution and incubate at 60° overnight. 2. Add 50 ~1 of well-suspended DNA capture reagent slurry to the sample, vortex vigorously, and place on a rotator for 30 min at room temperature. 3. Spin the tube for 30 sec in a microcentrifuge (12,000 g) to pellet the capture reagent-DNA complex. Carefully remove the supernatant with a micropipette and discard it appropriately (blood may be a biohazard). 4. Add 100/~1 of TE buffer, vortex vigorously, to wash the reagent and spin in a microcentrifuge for 30 sec. Carefully remove the supernatant with a micropipette and discard appropriately. 5. Repeat step 4 two more times. 6. To the pelleted capture reagent-DNA complex, add 50 /~1 of 0.1 M KOH. Vortex vigorously and place on a rotator for 10 min at room temperature. 7. Spin the reagent in a microcentrifuge for 30 sec. Carefully pipette the supernatant, containing the DNA, into a clean microcentrifuge tube. 8. To the supernatant, add 25 /xl of 7.5 M ammonium acetate and 200/zl of absolute ethanol. Incubate the sample on ice for 10 min; pellet the precipitated DNA in a microcentrifuge, dry the pellet in a vacuum centrifuge, and suspend it in 64/.d of distilled water. 9. Set up a 100-/zl polymerase chain reaction (PCR) (using all 64/zl of sample) as described in the instructions to the Cetus-Perkin Elmer (Norwalk, CT) Gene-Amp kit? Following the reaction, the PCR products are ethanol precipitated, suspended in 15/xl of TE buffer, electrophoresed in a 4% (w/v) horizontal agarose gel (5.7 x 8.3 cm in a Bethesda Research Laboratories Horizon 58 apparatus), and visualized by ethidium bromide staining.
Protocol 3: Isolation of DNA from M13 Phage Lysates and Analysis by DNA Sequencing 1. As described in Sambrook et al., 4 precipitate phage particles from 1.2 ml of a cleared supernatant from an M13-infected culture by adding 0.3 ml of 2.5 M NaCI containing 20% (w/v) polyethylene glycol (PEG 8OOO). 2. Incubate for 15 min at room temperature and spin the tube in a microcentrifuge for 10 min. Remove and discard the supernatant. 3 R. Saiki, D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis, and H. Erlich, Science 239, 487 (1988). 4 j. Sambrook, E. Fritsch, and T. Maniatis, "Molecular Cloning, A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.
[4]
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3. Resuspend the pelleted phage particles in 50/zl of TE buffer and add 40/A of DNA extraction buffer and 10/.d of proteinase K solution. Incubate for 60 min at 60°. 4. Add 100/xl of capture reagent slurry, vortex vigorously, and place on a rotator for 30 min at room temperature. 5. Proceed as in steps 3 through 6 of protocol 1, above. 6. To the supernatant, containing the eluted DNA (in 50/xl of 0.5 N NaOH), add 25 /zl of 7.5 M ammonium acetate and 200/.d of absolute ethanol. Incubate the sample on ice for 10 min, pellet the precipitated DNA in a microcentrifuge, dry the pellet in a vacuum centrifuge, and suspend it in 10/A of TE buffer. Use 1 to 5/zl for sequencing according to the particular procedure that is being used. Comments on Protocols
The protocols described above have worked consistently in our hands and can be used as starting points for other applications. The basic protocols can often be shortened even further by reducing the initial capture reagent binding and final alkali elution steps to 5 min each and by eliminating the TE buffer wash of the reagent after the initial binding step. Likewise, the proteinase K digestion of the sample can sometimes be reduced. The success of these modifications depends on the particular type of sample being assayed. For best results, two specific points should be kept in mind. First, the DNA capture reagent must be uniformly dispersed in the sample during the binding step. The investigator should use a rotator that turns the tubes completely end over end, rather than a shaking platform or other mixing device. Second, proteins in the sample must be digested thoroughly by proteinase K before the capture reagent is added to the sample. Use of the DNA extraction buffer helps assure efficient degradation of proteins. The capture reagent will bind undigested proteins, but not proteinase K digestion products. 1 Undigested proteins eluted from the reagent with the DNA can deleteriously affect dot-blot and sequencing assays. One potential limitation of the reagent should also be noted. Treatment with dilute alkali is the only effective means of releasing nucleic acid from the DNA capture reagent. Thus, we have analyzed the released DNA using procedures that do not require native DNA. Results
Basic Features of Capture and Release of DNA
We initially performed experiments to examine basic features of the capture and release protocols using radioactive DNA added to buffer or to
34
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[4]
human serum treated with proteinase K, as described in detail in Harding et al. 1 In summary these experiments indicated that (1) capture of DNA is independent of salt concentration up to at least 3 M NaCI or KCI; (2) capture is independent of EDTA concentration up to at least 0.5 M; (3) capture occurs in the presence of detergents such as 0.1 to 1% sodium dodecyl sulfate or 1% (v/v) Triton X-100; (4) relatively small amounts of DNA are captured from large sample volumes, e.g., 10 ng of DNA from 0.5 ml of sample; (5) relatively large amounts of DNA are captured from small volumes, e.g., 5 /~g of DNA from 30 /zl of sample; (6) DNA is undegraded by the capture and release protocols; and (7) RNA can also be captured, although the utility of this feature is obviated by the requirement for alkaline release of nucleic acid from the capture reagent.
Capture and Characterization of DNA from Complex Biological Samples Serum and Urine. Results of an experiment demonstrating capture and dot-blot quantitation of viral DNAs in serum or urine, as performed by protocol 1 above, are shown in Fig. 1. In the experiment (columns 1-4, Fig. 1), various amounts of a plasmid containing a cloned hepatitis B viral genome (HBV) were added to normal (uninfected) serum, captured, released, dot-blotted, and probed with an HBV RNA probe. As seen in Fig. 1 (columns 2 and 4, row c), 0.5 pg of the HBV target DNA was detected on nylon or nitrocellulose membranes, respectively. As controls, alkali-denatured plasmid was dot-blotted directly onto the filters in Fig. 1 (columns 1 and 3). Comparison of the intensities of the spots (measured by laser densitometry) in columns 1-4 in Fig. 1 indicate that the signal is about 30% as intense for the samples captured from serum as for the control samples spotted directly onto the filter. In row f of columns 2 and 4 (Fig. 1), the serum that was treated with capture reagent contained no added HBV DNA. The absence of signals in these rows indicates that proteins or other potential contaminants in the serum do not cause spurious signals on the dot-blot. The results shown in columns 7-9 of Fig. 1 indicate that HBV DNA present in virus particles can be captured and quantitated. The source of HBV-infected serum was a positive control from a commercially available HBV test kit (HepProbe; Life Technologies, Inc.). A hybridization signal was obtained from 50/zl of infected serum (Fig. 1, columns 8 and 9, row a) and from 50/zl of a 1 : 10 mixture of infected serum and normal serum (Fig. 1, columns 8 and 9, row b). Normal serum alone gave no signal (Fig. 1, columns 8 and 9, row e). Comparison of the intensity of the spots from
[4]
D N A ISOLATION USING METHIDIUM--SPERMINE--SEPHAROSE
12
34
56
35
789
a
b C
d e
~i~!
~ !!~iii~ii ii
.
~
~
~
~
~,~
/~
FIG. 1. Dot-blot analysis of viral DNAs captured from human serum or urine. Column 1: Standard amounts of plasmid pHBVT702 DNAs (containing a cloned hepatitis B virus sequence) were added to 200 p.l of 0.5 N NaOH and applied directly to a nylon membrane (Biotrans, Pall Corp., Glen Cove, NY). Column 2: Standard amounts of plasmid DNAs were added to normal serum, captured, and released as in protocol 1, applied to the filter, and hybridized with an HBV R N A probe. A 7-day exposure of the autoradiograph is shown. Columns 3 and 4: The same as columns 1 and 2, respectively, except that a nitrocellulose filter was used. A 4-day exposure o f the autoradiograph is shown. Row a, 50-pg HBV target; row b, 5-pg target; row c, 0.5-pg target; row d, 0.25-pg target; row e, 0.05-pg target; row f, no target. Column 5: As for column 1 except that plasmid pT7T3-19CMV DNAs (containing a cloned cytomegalovirus sequence) were applied to a Biotrans membrane. Column 6: Standard amounts of CMV plasmid DNAs captured from human urine. Row a, 67-pg target; row b, 6.7-pg target; row c, 0.67-pg target; row d, 0.33-pg target; row e, 0.067-pg target; row f, no target. The filter was hybridized with a CMV R N A probe; a 5-day exposure o f the autoradiograph is shown. Column 7: The same as column 1 (plasmid pHBVT702). Columns 8 and 9: Serum containing HBV virus (see text) was diluted with normal serum (where appropriate) and incubated with capture reagent as described in protocol 1. DNA released from the reagent was applied to a Biotrans membrane and hybridized with an HBV R N A probe. Row a, undiluted, infected serum; row b, infected serum diluted 1 : 10 with normal serum; row c, 1 : 100 dilution of infected serum; row d, 1 : 200 dilution of infected serum; row e, normal serum. A 7-day exposure of the autoradiograph is shown. Hybridization o f each filter was performed as follows: The filter was prehybridized for 30 min at 65 ° in 10% (v/v) formamide, 10% (w/v) dextran sulfate, 5 × SSPE (1 × SSPE: 0.18 M NaCI, 10 m M sodium phosphate, 1 m M Na2EDTA; pH 7.4), 5% (w/v) sodium dodecyl sulfate, and 100/zg/ml sheared herring sperm DNA. (Herring sperm D N A was not included when nylon membranes were used.) The filter was hybridized with 1 ml of hybridization solution (prehybridization solution containing 10 7 cpm of R N A probe) for 3 hr at 65 °. Following hybridization, the filter was washed briefly at room temperature with 2 x SSPE, incubated for 5 min at room temperature with 5/xg/ml RNase A in 2 × SSPE, and washed three times for 5 min in 0.1 × SSPE, 0.1% sodium dodecyl sulfate at 65 °. The filter was dried and autoradiographed at - 70 ° with two intensifying screens. (Data are reproduced from Harding et al) by permission of Oxford University Press.)
36
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
1
2
3
4
5
6
7
8
[4]
9
FIG. 2. Polymerase chain reaction analysis of DNA captured from whole blood and cultured cells. Human genomic DNA was isolated using the capture reagent either from whole blood or from a HeLa cell culture as in protocol 2. The polymerase chain reaction was performed using the GH18 and GH19 primers of Scharf et al., 5 complementary to specific human fl-globin gene sequences, for 30 cycles. The reaction products were electrophoresed on a 4% agarose gel as described in protocol 2. Ethidium bromide-stained gels are shown. Lane h The 123-bp ladder (Bethesda Research Laboratories) size markers. The fragment of greatest mobility is 123 bp in size. PCR reaction products are from DNA isolated from 10/zl of whole blood (lane 2), 1/xl of whole blood (lane 3), and from a control plasmid containing the fl-globin sequence (lane 4). Lane 5: 123-bp ladder size markers. PCR reaction products are from DNA isolated from 10,000 HeLa cells (lane 6), 1000 HeLa cells (lane 7), 100 HeLa cells (lane 8), and control plasmid (lane 9). Lanes 1-4 and 5-9, respectively, are from different gels. (Data are reproduced from Harding et al. 1by permission of Oxford University Press.)
t h e i n f e c t e d s e r u m w i t h t h e c o n t r o l s p o t s o f c o l u m n 7 in Fig. l , t o g e t h e r w i t h t h e r e s u l t o b t a i n e d a b o v e i n d i c a t i n g t h a t t h e h y b r i d i z a t i o n a s s a y is a b o u t 30% efficient, i n d i c a t e s t h a t 5 0 / z l o f t h e i n f e c t e d s e r u m c o n t a i n s a b o u t 15 p g o f H B V D N A . T h e e x p e r i m e n t in c o l u m n s 5 a n d 6 (Fig. l) i n d i c a t e s t h a t c y t o m e g a l o v i r u s D N A ( C M V ) a d d e d to u r i n e c a n a l s o b e d e t e c t e d u s i n g p r o t o c o l l , a b o v e . A p p r o x i m a t e l y 0.7 p g o f D N A w a s d e t e c t e d in t h e C M V - p o s i t i v e s a m p l e in c o l u m n 6, r o w c (Fig. 1), a n d signal w a s n o t o b t a i n e d f r o m u r i n e l a c k i n g C M V D N A (Fig. 1, c o l u m n 6, r o w f). W h o l e B l o o d a n d E u k a r y o t i c Cell Cultures. T h e r e s u l t s o f t h e e x p e r i m e n t in F i g . 2 d e m o n s t r a t e t h a t D N A c a n b e c a p t u r e d f r o m c u l t u r e d h u m a n c e l l s o r w h o l e b l o o d , a c c o r d i n g to p r o t o c o l 2 ( a b o v e ) , a n d c h a r a c t e r i z e d by the polymerase chain reaction (PCR). 5 Lanes 2 and 3 of Fig. 2 show that human fl-globin PCR reaction prod5 S. Scharf, G. Horn, and H. Erlich, Science 233, 1076 (1986).
[4]
DNA
ISOLATION USING METHIDIUM--SPERMINE--SEPHAROSE
37
ucts are readily detected from genomic DNA captured from 10 or 1/zl of whole blood, respectively. Lanes 6-8 of Fig. 2 show PCR reaction products obtained from I0,000, 1000, or 100 cultured human cells, respectively. For both experiments of Fig. 2, control reactions were performed in which template DNAs were not included in the PCR reactions. No PCR reaction products (including /3-globin products and "primer-dimers") were synthesized in these reactions, indicating that, as expected, the PCR was template dependent (data not shown). The PCR reactions of both experiments in Fig. 2 show relatively little dose response. This probably reflects two factors. First, the PCR reaction itself is not linear under the particular conditions used in these experiments. Second, to assure a gel band that reproduced well for publication, we applied the entire PCR reaction to the gel. If smaller amounts of reaction products are run on the gel, a nonlinear dose response is observed. M13 Phage Lysates. The results of Fig. 3 demonstrate that high-quality single-stranded phage M 13 sequencing templates are prepared using protocol 3 (above). The first four lanes on the left-hand side of Fig. 3 are sequencing reactions obtained from single-stranded M 13 DNA isolated by standard phenol extraction protocols 4 and the four lanes on the right-hand side of Fig. 3 are obtained from M13 DNA isolated using the capture reagent. Inspection of Fig. 3 indicates that the same sequence can be read from both sets of lanes, demonstrating the utility of capture reagent for preparing sequencing templates.
Conclusions The DNA capture reagent has two notable features that make it a useful alternative to traditional methods for DNA isolation. First is its ease of use. DNA is captured from serum or whole blood in a 45-min procedure that does not involve organic solvents. In addition, the reagent is amenable to batch processes. For example, 24 samples in microcentrifuge tubes (enough to fill a microcentrifuge rotor) can readily be analyzed simultaneously by one operator. The second important feature of the capture reagent is its versatility. It can be used to isolate DNA from many different biological samples. Capture is independent of the salt concentration of the sample and occurs in the presence of detergents, proteinase K, and EDTA, which are often used in protocols for the isolation of viral and cellular DNAs from complex samples. Very small amounts of DNA can be isolated from large volumes of sample and analyzed using a sensitive assay such as hybridization or the polymerase chain reaction.
38
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
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ACGTACGT
FIG. 3. Sequencing reactions performed on M13 DNA captured from cell lysates. MI3 DNA was isolated as in protocol 3. Sequencing reactions were performed using the KiloBase Sequencing System (Bethesda Research Laboratories) according to the manufacturer's instructions. Reaction products were resolved on a 6% polyacrylamide-8 M urea gel at 60 W in Tris-borate EDTA buffer. The first four lanes (left-hand side) are reactions using DNA isolated by standard phenol extraction procedures (Sambrook e t a l . ) . 4 The other four lanes (right-hand side) are reactions using DNA captured and released from the capture reagent. (Data are reproduced from Harding e t a L I by permission of Oxford University Press.)
[5]
AFFINITY CAPTURE FOR SELECTIVE ENRICHMENT OF D N A
39
These features should facilitate analysis of DNA from pathogens or other DNAs of interest in complex biological samples. Acknowledgments We thank our colleagues, Leonard Klevan and Dietmar Rabussay, for support and encouragement and Jim D'Alessio, Sherry Challberg, and Jim Hartley for providing plasmids and HBV-infected serum.
[5] C h r o m o s o m e Fishing: A n Affinity C a p t u r e M e t h o d for S e l e c t i v e E n r i c h m e n t o f Large G e n o m i c D N A F r a g m e n t s
By RAJENDRA P.
K A N D P A L , DAVID C. W A R D ,
and
SHERMAN M . WEISSMAN
Several technological advances have made it feasible to construct large-scale restriction maps of complex genomes. A combination of various techniques, i.e., pulsed-field gel electrophoresis (PFGE), 1-3 the construction of linking and jumping libraries,4-7 and cosmid 8 and yeast artificial chromosome (YAC) 9 cloning and selection methods, is available to expedite genome mapping. The isolation of specific sequences from a plasmid library also has been achieved by avidin-biotin affinity chromatography after a RecA-mediated search for homologous sequences ~° by an appropriate probe.~l Despite the advantages these methods provide, there have been technical difficulties and limitations in their application. Conventional methods of mapping involve the isolation of end probes from plasmid or phage clones and successive screening of libraries to obtain overlapping J D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 67 (1984). 2 G. F. Carle, M. Frank, and M. V. Olson, Science 232, 65 (1986). 3 G. Chu, D. Vollrath, and R. W. Davis, Science 234, 1982 (1986). 4 C. L. Smith, S. K. Lawrance, G. A. GiUespie, C. R. Cantor, S. M. Weissman, and F. S. Collins, this series, Vol. 151, p. 461. 5 M. R. Wallace, J. W. Fountain, A. M. Bereton, and F. S. Collins, Nucleic Acids Res. 17, 1655 (1989). 6 F. S. Collins and S. M. Weissman, Proc. Natl. Acad. Sci. U.S.A. 81, 6812 (1984). 7 A. M. Poustaka and H. Lehrach, Trends Genet. 2, 174 (1986). 8 j. Collins and B. Hohn, Proc. Natl. Acad. Sci. U.S.A. 75, 4242 (1978). 9 D. T. Burke, G. F. Carle, and M. V. Olson, Science 235, 806 (1987). to B. Rigas, A. A. Welcher, D. C. Ward, and S. M. Weissman, Proc. Natl. Acad. Sci. U.S.A. 83, 9591 (1986). H C. M. Radding, Annu. Rev. Genet. 16, 405 (1986).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright© 1992by AcademicPress. Inc. All rightsof reproductionin any form reserved.
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5. Add 30 ~g yeast tRNA per milliliter of D N A solution. 24 Add 100/zg of proteinase K and incubate at 50 ° for I to 2 hr. Extract twice with equal volumes of a 1 : 1 mixture of phenol-chloroform,18 and once with an equal volume of chloroform alone. Separate the layers by centrifugation at full speed in a microfuge at room temperature, saving the aqueous layer at each stage for the subsequent step. 6. Following these organic extractions, transfer the aqueous layer to a 1.5-ml microfuge tube. Add two volumes of ethanol and mix well. Store at - 70 ° for 30 min. Spin in a microfuge at 4 ° for 15 min to pellet down the D N A precipitate. 7. Resuspend the pellet in 400 /zl of T E containing 0.3 M sodium acetate (pH 7.0), and precipitate the D N A again, by mixing with 2 vol of ethanol. Store at - 7 0 ° for 30 min. Spin in a microfuge at 4 ° for 15 min to pellet down the D N A precipitate. 8. Dissolve the pellet in 40/xl TE. The solution contains low molecular weight D N A along with some RNA. The D N A preparation is suitable for diagnostic restriction digestions followed by analysis involving Southern blot hybridization using a labeled probe specific for the DNA. 25 If the isolated low molecular weight D N A is a bacterial plasmid, this D N A preparation is also suitable for reintroduction into bacteria by transformation. 12 Acknowledgment We thank Julie Yamaguchi and Daniel C. Alter for help with standardization of the boiling method and development of the phenol-chloroform lysis-extraction method, respectively, and Dr. Cho-Yau Yeung for helpful discussions. We thank Linda Cardenas for clerical assistance. This work was supported by grants from the National Institutes of Health, the American Cancer Society, and the University of Illinois at Chicago. 24The tRNA is added as a carrier at this stage to minimize loss of DNA during the organic extractions and to help in the precipitation of the DNA at later steps. 25E. M. Southern, J. Mol. Biol. 98, 503 (1975).
[4] D N A I s o l a t i o n U s i n g M e t h i d i u m - S p e r m i n e - S e p h a r o s e B y JOHN D. HARDING, ROBERT L. BEBEE, and GULILAT GEBEYEHU
Introduction Rapid, quantitative isolation of D N A from complex biological samples is required for many protocols in molecular biology and molecular diagnosMETHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[4]
tics. Standard methods using organic solvents such as phenol are reliable, but are time consuming and involve the use of toxic chemicals. Therefore many alternative methods for DNA isolation have been examined over the past several years. In designing novel reagents for rapid isolation of DNA from complex samples, we reasoned that a very efficient "capture reagent" would consist of an intercalating dye attached to a solid support by a molecular linker.l'2 The intercalator binds the DNA with high affinity and the solid support allows rapid separation of the bound material from unwanted contaminants. In this chapter we describe protocols for isolating DNA from a variety of complex samples using a DNA capture reagent consisting of the intercalator, methidium, attached to a Sepharose bead by a spermine linker. DNA is released from the reagent in 0.1 to 0.5 N KOH or NaOH and is characterized by procedures such as dot-blot hybridization, sequencing, or polymerase chain reaction analysis. Materials DNA capture reagent (methidium-spermine-Sepharose) and DNA extraction buffer [Cat. No. 80885A; Bethesda Research Laboratories (Life Technologies, Inc., Gaithersburg, MD)]: The DNA capture reagent is synthesized as described in Harding et al.l Proteinase K solution: 50 mg/ml proteinase K (Bethesda Research Laboratories) in 0.2 M Tris-HCl, 0.1 M NazEDTA, 3% (v/v) Brij 35; pH 7.5 TE buffer: 10 mM Tris-HC1, 1 mM Na2EDTA; pH 7.5
Methods Protocol 1: Isolation o f D N A from Serum or Urine and Characterization by Dot-Blot Analysis 1. To 50/xl of serum or urine in a 1.5-ml microcentrifuge tube add 40/zl of DNA extraction buffer and 10/xl of proteinase K solution. Incubate for 60 min at 65 °. 2. Vortex the stock tube of DNA capture reagent vigorously for a few seconds to suspend the capture reagent uniformly and pipette 50/xl of the slurry into the sample tube. Vortex the sample tube vigorously for a few i j. Harding, G. Gebeyehu, R. Bebee, D. Simms, and L. Klevan, Nucleic Acids Res. 17, 6947 (1989). 2 G. Gebeyehu, L. Klevan, and J. Harding, U.S. Patent 4,921,805 (1990).
[4]
DNA ISOLATIONUSINGMETHIDIUM--SPERMINE-SEPHAROSE
31
seconds and immediately place it on a rotator (such as a Labquake rotator, Labindustries Inc., Berkeley, CA) for 30 min at room temperature. 3. Spin the tube for 30 sec in a microcentrifuge (12,000 g) to pellet the capture reagent-DNA complex. Carefully remove the supernatant with a micropipette and discard it appropriately (serum and urine may be biohazards). 4. Add 100/~1 of TE buffer, vortex vigorously, and spin in a microcentrifuge for 30 sec. Carefully remove the supernatant with a micropipette and discard appropriately. 5. To the capture reagent pellet add 100 ~1 of 0.5 N NaOH. Vortex vigorously for a few seconds and place on a rotator for 10 min at room temperature. 6. Spin out the capture reagent in a microcentrifuge for 30 sec and carefully remove the supernatant, which contains the DNA, with a micropipette. 7. For alkaline dot blotting, presoak the membrane (nitrocellulose or nylon) in 0.5 N NaOH for 5 min with gentle agitation. Place the membrane on a dot-blot apparatus. Pass the sample, eluted directly from the capture reagent, through the membrane. Wash each dot-blotted sample with 500/~1 of 0.5 N NaOH. For nitrocellulose, bake the membrane for 1 hr at 80° in a vacuum oven. For nylon, treat the membrane according to the instructions of the manufacturer. Prehybridize and hybridize the membrane by standard protocols or using conditions previously optimized for a particular probe. 8. An alternative dot-blotting technique that has worked equally well is as follows. To the sample eluted from the capture reagent, add an equal volume of 2 M ammonium acetate. Ammonium acetate is prepared by dissolving the solid salt to a 2 M final concentration; the pH is not adjusted. Before applying the sample, soak the membrane briefly in distilled water and then for 5 min in 1 M ammonium acetate with gentle agitation. Place the membrane on a dot-blot apparatus. Pass the DNA sample through the membrane; wash each dot blotted sample with 500/xl of I M ammonium acetate. If the membrane is to be baked prior to hybridization (e.g., nitrocellulose and some nylons), incubate it in 20 × SSC (3 M NaCI, 0.3 sodium citrate, pH 7.0) with gentle agitation for 5 min at room temperature prior to the baking step. Protocol 2: Isolation of DNA from Whole Blood or Cultured Cells and Analysis by Polymerase Chain Reaction 1. Dilute 1 to 10/~1 of whole blood, collected in ethylenediaminetetraacetic acid (EDTA) as an anticoagulant, to a total volume of 50/xl with TE buffer in a 1.5-ml microcentrifuge tube. If cultured cells are to be
32
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[4]
analyzed, pellet the cells from the culture medium and resuspend the pellet in 50/xl of TE buffer. To either sample, add 40/xl of DNA extraction buffer and 10/~1 of proteinase K solution and incubate at 60° overnight. 2. Add 50 ~1 of well-suspended DNA capture reagent slurry to the sample, vortex vigorously, and place on a rotator for 30 min at room temperature. 3. Spin the tube for 30 sec in a microcentrifuge (12,000 g) to pellet the capture reagent-DNA complex. Carefully remove the supernatant with a micropipette and discard it appropriately (blood may be a biohazard). 4. Add 100/~1 of TE buffer, vortex vigorously, to wash the reagent and spin in a microcentrifuge for 30 sec. Carefully remove the supernatant with a micropipette and discard appropriately. 5. Repeat step 4 two more times. 6. To the pelleted capture reagent-DNA complex, add 50 /~1 of 0.1 M KOH. Vortex vigorously and place on a rotator for 10 min at room temperature. 7. Spin the reagent in a microcentrifuge for 30 sec. Carefully pipette the supernatant, containing the DNA, into a clean microcentrifuge tube. 8. To the supernatant, add 25 /xl of 7.5 M ammonium acetate and 200/zl of absolute ethanol. Incubate the sample on ice for 10 min; pellet the precipitated DNA in a microcentrifuge, dry the pellet in a vacuum centrifuge, and suspend it in 64/.d of distilled water. 9. Set up a 100-/zl polymerase chain reaction (PCR) (using all 64/zl of sample) as described in the instructions to the Cetus-Perkin Elmer (Norwalk, CT) Gene-Amp kit? Following the reaction, the PCR products are ethanol precipitated, suspended in 15/xl of TE buffer, electrophoresed in a 4% (w/v) horizontal agarose gel (5.7 x 8.3 cm in a Bethesda Research Laboratories Horizon 58 apparatus), and visualized by ethidium bromide staining.
Protocol 3: Isolation of DNA from M13 Phage Lysates and Analysis by DNA Sequencing 1. As described in Sambrook et al., 4 precipitate phage particles from 1.2 ml of a cleared supernatant from an M13-infected culture by adding 0.3 ml of 2.5 M NaCI containing 20% (w/v) polyethylene glycol (PEG 8OOO). 2. Incubate for 15 min at room temperature and spin the tube in a microcentrifuge for 10 min. Remove and discard the supernatant. 3 R. Saiki, D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis, and H. Erlich, Science 239, 487 (1988). 4 j. Sambrook, E. Fritsch, and T. Maniatis, "Molecular Cloning, A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.
[4]
DNA ISOLATIONUSINGMETHIDIUM--SPERMINE--SEPHAROSE
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3. Resuspend the pelleted phage particles in 50/zl of TE buffer and add 40/A of DNA extraction buffer and 10/.d of proteinase K solution. Incubate for 60 min at 60°. 4. Add 100/xl of capture reagent slurry, vortex vigorously, and place on a rotator for 30 min at room temperature. 5. Proceed as in steps 3 through 6 of protocol 1, above. 6. To the supernatant, containing the eluted DNA (in 50/xl of 0.5 N NaOH), add 25 /zl of 7.5 M ammonium acetate and 200/.d of absolute ethanol. Incubate the sample on ice for 10 min, pellet the precipitated DNA in a microcentrifuge, dry the pellet in a vacuum centrifuge, and suspend it in 10/A of TE buffer. Use 1 to 5/zl for sequencing according to the particular procedure that is being used. Comments on Protocols
The protocols described above have worked consistently in our hands and can be used as starting points for other applications. The basic protocols can often be shortened even further by reducing the initial capture reagent binding and final alkali elution steps to 5 min each and by eliminating the TE buffer wash of the reagent after the initial binding step. Likewise, the proteinase K digestion of the sample can sometimes be reduced. The success of these modifications depends on the particular type of sample being assayed. For best results, two specific points should be kept in mind. First, the DNA capture reagent must be uniformly dispersed in the sample during the binding step. The investigator should use a rotator that turns the tubes completely end over end, rather than a shaking platform or other mixing device. Second, proteins in the sample must be digested thoroughly by proteinase K before the capture reagent is added to the sample. Use of the DNA extraction buffer helps assure efficient degradation of proteins. The capture reagent will bind undigested proteins, but not proteinase K digestion products. 1 Undigested proteins eluted from the reagent with the DNA can deleteriously affect dot-blot and sequencing assays. One potential limitation of the reagent should also be noted. Treatment with dilute alkali is the only effective means of releasing nucleic acid from the DNA capture reagent. Thus, we have analyzed the released DNA using procedures that do not require native DNA. Results
Basic Features of Capture and Release of DNA
We initially performed experiments to examine basic features of the capture and release protocols using radioactive DNA added to buffer or to
34
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[4]
human serum treated with proteinase K, as described in detail in Harding et al. 1 In summary these experiments indicated that (1) capture of DNA is independent of salt concentration up to at least 3 M NaCI or KCI; (2) capture is independent of EDTA concentration up to at least 0.5 M; (3) capture occurs in the presence of detergents such as 0.1 to 1% sodium dodecyl sulfate or 1% (v/v) Triton X-100; (4) relatively small amounts of DNA are captured from large sample volumes, e.g., 10 ng of DNA from 0.5 ml of sample; (5) relatively large amounts of DNA are captured from small volumes, e.g., 5 /~g of DNA from 30 /zl of sample; (6) DNA is undegraded by the capture and release protocols; and (7) RNA can also be captured, although the utility of this feature is obviated by the requirement for alkaline release of nucleic acid from the capture reagent.
Capture and Characterization of DNA from Complex Biological Samples Serum and Urine. Results of an experiment demonstrating capture and dot-blot quantitation of viral DNAs in serum or urine, as performed by protocol 1 above, are shown in Fig. 1. In the experiment (columns 1-4, Fig. 1), various amounts of a plasmid containing a cloned hepatitis B viral genome (HBV) were added to normal (uninfected) serum, captured, released, dot-blotted, and probed with an HBV RNA probe. As seen in Fig. 1 (columns 2 and 4, row c), 0.5 pg of the HBV target DNA was detected on nylon or nitrocellulose membranes, respectively. As controls, alkali-denatured plasmid was dot-blotted directly onto the filters in Fig. 1 (columns 1 and 3). Comparison of the intensities of the spots (measured by laser densitometry) in columns 1-4 in Fig. 1 indicate that the signal is about 30% as intense for the samples captured from serum as for the control samples spotted directly onto the filter. In row f of columns 2 and 4 (Fig. 1), the serum that was treated with capture reagent contained no added HBV DNA. The absence of signals in these rows indicates that proteins or other potential contaminants in the serum do not cause spurious signals on the dot-blot. The results shown in columns 7-9 of Fig. 1 indicate that HBV DNA present in virus particles can be captured and quantitated. The source of HBV-infected serum was a positive control from a commercially available HBV test kit (HepProbe; Life Technologies, Inc.). A hybridization signal was obtained from 50/zl of infected serum (Fig. 1, columns 8 and 9, row a) and from 50/zl of a 1 : 10 mixture of infected serum and normal serum (Fig. 1, columns 8 and 9, row b). Normal serum alone gave no signal (Fig. 1, columns 8 and 9, row e). Comparison of the intensity of the spots from
[4]
D N A ISOLATION USING METHIDIUM--SPERMINE--SEPHAROSE
12
34
56
35
789
a
b C
d e
~i~!
~ !!~iii~ii ii
.
~
~
~
~
~,~
/~
FIG. 1. Dot-blot analysis of viral DNAs captured from human serum or urine. Column 1: Standard amounts of plasmid pHBVT702 DNAs (containing a cloned hepatitis B virus sequence) were added to 200 p.l of 0.5 N NaOH and applied directly to a nylon membrane (Biotrans, Pall Corp., Glen Cove, NY). Column 2: Standard amounts of plasmid DNAs were added to normal serum, captured, and released as in protocol 1, applied to the filter, and hybridized with an HBV R N A probe. A 7-day exposure of the autoradiograph is shown. Columns 3 and 4: The same as columns 1 and 2, respectively, except that a nitrocellulose filter was used. A 4-day exposure o f the autoradiograph is shown. Row a, 50-pg HBV target; row b, 5-pg target; row c, 0.5-pg target; row d, 0.25-pg target; row e, 0.05-pg target; row f, no target. Column 5: As for column 1 except that plasmid pT7T3-19CMV DNAs (containing a cloned cytomegalovirus sequence) were applied to a Biotrans membrane. Column 6: Standard amounts of CMV plasmid DNAs captured from human urine. Row a, 67-pg target; row b, 6.7-pg target; row c, 0.67-pg target; row d, 0.33-pg target; row e, 0.067-pg target; row f, no target. The filter was hybridized with a CMV R N A probe; a 5-day exposure o f the autoradiograph is shown. Column 7: The same as column 1 (plasmid pHBVT702). Columns 8 and 9: Serum containing HBV virus (see text) was diluted with normal serum (where appropriate) and incubated with capture reagent as described in protocol 1. DNA released from the reagent was applied to a Biotrans membrane and hybridized with an HBV R N A probe. Row a, undiluted, infected serum; row b, infected serum diluted 1 : 10 with normal serum; row c, 1 : 100 dilution of infected serum; row d, 1 : 200 dilution of infected serum; row e, normal serum. A 7-day exposure of the autoradiograph is shown. Hybridization o f each filter was performed as follows: The filter was prehybridized for 30 min at 65 ° in 10% (v/v) formamide, 10% (w/v) dextran sulfate, 5 × SSPE (1 × SSPE: 0.18 M NaCI, 10 m M sodium phosphate, 1 m M Na2EDTA; pH 7.4), 5% (w/v) sodium dodecyl sulfate, and 100/zg/ml sheared herring sperm DNA. (Herring sperm D N A was not included when nylon membranes were used.) The filter was hybridized with 1 ml of hybridization solution (prehybridization solution containing 10 7 cpm of R N A probe) for 3 hr at 65 °. Following hybridization, the filter was washed briefly at room temperature with 2 x SSPE, incubated for 5 min at room temperature with 5/xg/ml RNase A in 2 × SSPE, and washed three times for 5 min in 0.1 × SSPE, 0.1% sodium dodecyl sulfate at 65 °. The filter was dried and autoradiographed at - 70 ° with two intensifying screens. (Data are reproduced from Harding et al) by permission of Oxford University Press.)
36
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
1
2
3
4
5
6
7
8
[4]
9
FIG. 2. Polymerase chain reaction analysis of DNA captured from whole blood and cultured cells. Human genomic DNA was isolated using the capture reagent either from whole blood or from a HeLa cell culture as in protocol 2. The polymerase chain reaction was performed using the GH18 and GH19 primers of Scharf et al., 5 complementary to specific human fl-globin gene sequences, for 30 cycles. The reaction products were electrophoresed on a 4% agarose gel as described in protocol 2. Ethidium bromide-stained gels are shown. Lane h The 123-bp ladder (Bethesda Research Laboratories) size markers. The fragment of greatest mobility is 123 bp in size. PCR reaction products are from DNA isolated from 10/zl of whole blood (lane 2), 1/xl of whole blood (lane 3), and from a control plasmid containing the fl-globin sequence (lane 4). Lane 5: 123-bp ladder size markers. PCR reaction products are from DNA isolated from 10,000 HeLa cells (lane 6), 1000 HeLa cells (lane 7), 100 HeLa cells (lane 8), and control plasmid (lane 9). Lanes 1-4 and 5-9, respectively, are from different gels. (Data are reproduced from Harding et al. 1by permission of Oxford University Press.)
t h e i n f e c t e d s e r u m w i t h t h e c o n t r o l s p o t s o f c o l u m n 7 in Fig. l , t o g e t h e r w i t h t h e r e s u l t o b t a i n e d a b o v e i n d i c a t i n g t h a t t h e h y b r i d i z a t i o n a s s a y is a b o u t 30% efficient, i n d i c a t e s t h a t 5 0 / z l o f t h e i n f e c t e d s e r u m c o n t a i n s a b o u t 15 p g o f H B V D N A . T h e e x p e r i m e n t in c o l u m n s 5 a n d 6 (Fig. l) i n d i c a t e s t h a t c y t o m e g a l o v i r u s D N A ( C M V ) a d d e d to u r i n e c a n a l s o b e d e t e c t e d u s i n g p r o t o c o l l , a b o v e . A p p r o x i m a t e l y 0.7 p g o f D N A w a s d e t e c t e d in t h e C M V - p o s i t i v e s a m p l e in c o l u m n 6, r o w c (Fig. 1), a n d signal w a s n o t o b t a i n e d f r o m u r i n e l a c k i n g C M V D N A (Fig. 1, c o l u m n 6, r o w f). W h o l e B l o o d a n d E u k a r y o t i c Cell Cultures. T h e r e s u l t s o f t h e e x p e r i m e n t in F i g . 2 d e m o n s t r a t e t h a t D N A c a n b e c a p t u r e d f r o m c u l t u r e d h u m a n c e l l s o r w h o l e b l o o d , a c c o r d i n g to p r o t o c o l 2 ( a b o v e ) , a n d c h a r a c t e r i z e d by the polymerase chain reaction (PCR). 5 Lanes 2 and 3 of Fig. 2 show that human fl-globin PCR reaction prod5 S. Scharf, G. Horn, and H. Erlich, Science 233, 1076 (1986).
[4]
DNA
ISOLATION USING METHIDIUM--SPERMINE--SEPHAROSE
37
ucts are readily detected from genomic DNA captured from 10 or 1/zl of whole blood, respectively. Lanes 6-8 of Fig. 2 show PCR reaction products obtained from I0,000, 1000, or 100 cultured human cells, respectively. For both experiments of Fig. 2, control reactions were performed in which template DNAs were not included in the PCR reactions. No PCR reaction products (including /3-globin products and "primer-dimers") were synthesized in these reactions, indicating that, as expected, the PCR was template dependent (data not shown). The PCR reactions of both experiments in Fig. 2 show relatively little dose response. This probably reflects two factors. First, the PCR reaction itself is not linear under the particular conditions used in these experiments. Second, to assure a gel band that reproduced well for publication, we applied the entire PCR reaction to the gel. If smaller amounts of reaction products are run on the gel, a nonlinear dose response is observed. M13 Phage Lysates. The results of Fig. 3 demonstrate that high-quality single-stranded phage M 13 sequencing templates are prepared using protocol 3 (above). The first four lanes on the left-hand side of Fig. 3 are sequencing reactions obtained from single-stranded M 13 DNA isolated by standard phenol extraction protocols 4 and the four lanes on the right-hand side of Fig. 3 are obtained from M13 DNA isolated using the capture reagent. Inspection of Fig. 3 indicates that the same sequence can be read from both sets of lanes, demonstrating the utility of capture reagent for preparing sequencing templates.
Conclusions The DNA capture reagent has two notable features that make it a useful alternative to traditional methods for DNA isolation. First is its ease of use. DNA is captured from serum or whole blood in a 45-min procedure that does not involve organic solvents. In addition, the reagent is amenable to batch processes. For example, 24 samples in microcentrifuge tubes (enough to fill a microcentrifuge rotor) can readily be analyzed simultaneously by one operator. The second important feature of the capture reagent is its versatility. It can be used to isolate DNA from many different biological samples. Capture is independent of the salt concentration of the sample and occurs in the presence of detergents, proteinase K, and EDTA, which are often used in protocols for the isolation of viral and cellular DNAs from complex samples. Very small amounts of DNA can be isolated from large volumes of sample and analyzed using a sensitive assay such as hybridization or the polymerase chain reaction.
38
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
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ACGTACGT
FIG. 3. Sequencing reactions performed on M13 DNA captured from cell lysates. MI3 DNA was isolated as in protocol 3. Sequencing reactions were performed using the KiloBase Sequencing System (Bethesda Research Laboratories) according to the manufacturer's instructions. Reaction products were resolved on a 6% polyacrylamide-8 M urea gel at 60 W in Tris-borate EDTA buffer. The first four lanes (left-hand side) are reactions using DNA isolated by standard phenol extraction procedures (Sambrook e t a l . ) . 4 The other four lanes (right-hand side) are reactions using DNA captured and released from the capture reagent. (Data are reproduced from Harding e t a L I by permission of Oxford University Press.)
[5]
AFFINITY CAPTURE FOR SELECTIVE ENRICHMENT OF D N A
39
These features should facilitate analysis of DNA from pathogens or other DNAs of interest in complex biological samples. Acknowledgments We thank our colleagues, Leonard Klevan and Dietmar Rabussay, for support and encouragement and Jim D'Alessio, Sherry Challberg, and Jim Hartley for providing plasmids and HBV-infected serum.
[5] C h r o m o s o m e Fishing: A n Affinity C a p t u r e M e t h o d for S e l e c t i v e E n r i c h m e n t o f Large G e n o m i c D N A F r a g m e n t s
By RAJENDRA P.
K A N D P A L , DAVID C. W A R D ,
and
SHERMAN M . WEISSMAN
Several technological advances have made it feasible to construct large-scale restriction maps of complex genomes. A combination of various techniques, i.e., pulsed-field gel electrophoresis (PFGE), 1-3 the construction of linking and jumping libraries,4-7 and cosmid 8 and yeast artificial chromosome (YAC) 9 cloning and selection methods, is available to expedite genome mapping. The isolation of specific sequences from a plasmid library also has been achieved by avidin-biotin affinity chromatography after a RecA-mediated search for homologous sequences ~° by an appropriate probe.~l Despite the advantages these methods provide, there have been technical difficulties and limitations in their application. Conventional methods of mapping involve the isolation of end probes from plasmid or phage clones and successive screening of libraries to obtain overlapping J D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 67 (1984). 2 G. F. Carle, M. Frank, and M. V. Olson, Science 232, 65 (1986). 3 G. Chu, D. Vollrath, and R. W. Davis, Science 234, 1982 (1986). 4 C. L. Smith, S. K. Lawrance, G. A. GiUespie, C. R. Cantor, S. M. Weissman, and F. S. Collins, this series, Vol. 151, p. 461. 5 M. R. Wallace, J. W. Fountain, A. M. Bereton, and F. S. Collins, Nucleic Acids Res. 17, 1655 (1989). 6 F. S. Collins and S. M. Weissman, Proc. Natl. Acad. Sci. U.S.A. 81, 6812 (1984). 7 A. M. Poustaka and H. Lehrach, Trends Genet. 2, 174 (1986). 8 j. Collins and B. Hohn, Proc. Natl. Acad. Sci. U.S.A. 75, 4242 (1978). 9 D. T. Burke, G. F. Carle, and M. V. Olson, Science 235, 806 (1987). to B. Rigas, A. A. Welcher, D. C. Ward, and S. M. Weissman, Proc. Natl. Acad. Sci. U.S.A. 83, 9591 (1986). H C. M. Radding, Annu. Rev. Genet. 16, 405 (1986).
METHODS IN ENZYMOLOGY,VOL. 216
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