- Email: [email protected]
[19]Chemical cleavage of mismatch to detect mutations
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MUTAGENESIS AND GENE DISRUPTION
[19]
J16, although the latter has a lower AG by 7.5 kcal, there was no difference in the frequency at which mutations were obtained. The methodology for carrying out site-directed mutagenesis has evolved greatly over the past few years. Beyond techniques based on similar methodology as described here, the most significant advance has been in the application of the polymerase chain reaction (PCR) to generate site-directed mutations. A number of protocols have been developed using either two or three oligonucleotide primers. Due to the practical limits of the PCR using Taq DNA polymerase, only a short region is amplified and then cloned into a recipient vector. One potential problem, which has not been extensively documented, is the known lack of fidelity of these thermostable DNA polymerases, especially Taq DNA polymerase, which may introduce second-site mutations in the target. Acknowledgments The authors acknowledge the contributions of Clara Finch, Jonathan Miller, Parke Flick, and Jennifer Webb for supplying data on the mutation frequencies. This work was supported by a grant from the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, the U.S. Army Research Office, and the National Science Foundation.
[19] C h e m i c a l C l e a v a g e o f M i s m a t c h to D e t e c t M u t a t i o n s By JENNIFER A. SALEEBA and RICHARD G. H. COTTON Introduction The chemical cleavage of mismatch (CCM) method allows mutation sites to be detected in kilobase-length pieces of nucleic acids. J A screening method such as this obviates the need to sequence lengths of DNA to determine mutation sites. The method can be widely applied. It can be used to compare a clone of known or unknown sequence to samples of mutant origin, and for applications such as confirmation of in vitro mutagenesis. It has been
J R. G. H. Cotton, N. R. Rodrigues, and D. R. Campbell, Proc. Natl. Acad. Sci. U.S.A. 85, 4397 (1988).
METHODS IN ENZYMOLOGY.VOL. 217
Copyright© 1993by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[19]
CHEMICAL CLEAVAGE OF MISMATCH METHOD
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widely applied to diagnosis of human inherited disease 2-7 and to mutation detection in other organisms. 8-1°
Principle of the Chemical Cleavage Method Chemical cleavage is based on the principle that mismatched or unmatched residues are more reactive to modification by the chemicals hydroxylamine and osmium tetroxide than matched bases. This can be used to advantage for detection of point mutations and small insertions and deletions. Heteroduplex molecules can be formed by annealing a control and a test piece of DNA. Sites at which residues are mismatched or unmatched will be modified by the chemical hydroxylamine, in the case of cytosine residues, and osmium tetroxide, in the case of thymine residues. Cleavage of the DNA at the site of the modified residues is achieved by subsequent reaction with piperidine. Cleavage products are resolved by denaturing gel electrophoresis, and detection usually takes place by autoradiography (Fig. I). If necessary the exact change present in a mutant is then determined by sequencing the region identified by CCM. Mismatched or unmatched guanine and adenine residues are detected by virtue of the fact that their complementary cytosine and thymine residues are reactive. Cytosine and thymine residues surrounding mismatch sites may have a limited reactivity with hydroxylamine and osmium tetroxide, due to disruption of the helix.11 Analysis of a number of DNA fragments screened for mutations by chemical cleavage has shown that some T residues in T. G mismatches are resistant to modification by osmium tetroxide, and subsequent piperidine 2 j. F. Bateman, S. R. Lamande, H.-H. M., Dahl, D. Chan, T. Mascara, and W. G. Cole, J. Biol. Chem. 264, 10960 (1989). 3 M. Grompe, C. T. Muzny, and C. T. Caskey, Proc. Natl. Acad. Sci. U.S.A. 86, 5888 (1989). 4 A. J. Montandon, P. M. Green, F. Giannelli, and D. R. Bentley, Nucleic Acids Res, 17, 3347 (1989). 5 D. W. Howells, S. M. Forrest, H.-H. M. Dahl, and R. G. H. Cotton, Am. J. Hum. Genet. 47, 279 (1990). I. Dianzani, S, M. Forrest, C. Camaschella, G. Saglio, A. Ponzone, and R. G. H. Cotton, Am. J. Hum. Genet. 48, 631 (1991). 7 S. M. Forrest, H.-H. M. Dahl, D. W. Howells, I. Dianzani, and R. G. H. Cotton, Am. J. Hum. Genet. 49, 175 (1991). 8 R. G. H. Cotton and P. J. Wright, J. Virol. Methods 26, 67 (1989). 9 M. Han and P. W. Steinberg, Cell 63, 921 (1990). l0 M. Grompe, J. Versalovic, T. Koeuth, and J. R. Lupski, J. Bacteriol. 173, 1268 (1991). tl R. G. H. Cotton and R. D. Campbell, Nucleic Acids Res. 17, 4223 (1989).
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--T --A
G C
--G ----C
[19]
C G,
I formation Heteroduplex -~C
#
G
~A
C#--'~
I Cleavage H
OT
FIG. 1. Principle behind chemical cleavage. Wild-type and mutant DNAs are heated and annealed to give heteroduplex molecules. Mismatched T resides are reactive to osmium tetroxide (*) and mismatched C residues are reactive to hydroxylamine (#). Cleavage of DNA strands occurs at these sites by reaction with piperidine. Cleavage products are resolved by denaturing gel electrophoresis, and detected by autoradiography or silver staining. A probe band in the first lane and cleavage products after reaction with hydroxylamine (H) and osmium tetroxide (0T) are indicated.
cleavage. 12 The sequence context surrounding the mismatch appears to be important in determining whether a particular T. G mismatch will be modified by osmium tetroxide. Fortunately, a heteroduplex containing a T. G mismatch is always accompanied by a complementary heteroduplex containing an A. C mismatch. An unreactive T- G mismatch can therefore be easily detected by the complementary A. C mismatch, which is highly reactive to hydroxylamine. With this approach, a strategy offering complete detection of mutations by chemical cleavage can be achieved. When using radiolabeled detection, probes are made from both test and control DNAs and used either separately or together in CCM reactions. If using 12 R. G. H. Cotton, manuscript in preparation.
[19]
CHEMICAL CLEAVAGE OF M1SMATCH METHOD
289
a nonradioactive mode, where all DNA is detected, one set of CCM reactions will allow all mutations to be identified. Internally and end-radiolabeled probes and unlabeled DNA may be used to detect cleavage products in CCM. End-labeled probes will be cleaved to reveal one detectable cleavage product for each mutation. This type of probe is best applied to examples in which a number of mutations are expected. If the exact position of the mutation must be known, a single strand should be labeled. Internally radiolabeled probes will give two cleavage products per mutation. These probes are easy to prepare and are therefore well suited to mutation detection in a number of samples in which few or no mutations are expected. Radiolabeled probes made from control and test DNAs must be included either together in one experiment, or separately in a series of experiments, to ensure detection of any unreactive T. G mismatches by their complementary A. C mismatch and completely screen a region of DNA for mutations. Unlabeled DNA may also be used, in which case cleavage products can be detected by methods such as silver staining. All DNA is identified in experiments performed in this way, and therefore complete mutation screening is assured. A variety of nucleic acid templates may be used in chemical cleavage. Cloned fragments, polymerase chain reaction (PCR)-amplified fragments, cDNA, and RNA have all been successfully applied to the identification of mutations.
Materials and Reagents Annealing buffer (2 x ): 1.2 M NaCI, 12 mM Tris-HCl (pH 7.5), 14 mM MgCi2; store at room temperature Formamide annealing buffer: 80% formamide, 40 mM piperazine-N, N'bis(2-ethanesulfonic acid) (PIPES) (pH 6.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.4 M NaC1; store at - 2 0 ° Hydroxylamine solution: Dissolve 1.39 g hydroxylamine hydrochloride or hydroxylammonium chloride (BDH, Poole, England) in 1.6 ml warm distilled HEO in glass. Add 1.75 ml diethylamine dropwise. Final pH should be 6.0. Store at 4° for up to 2 months Osmium tetroxide buffer (10x): 100 mM Tris-HCl (pH 7.7), I0 mM EDTA, 15% (v/v) pyridine (Sigma, St. Louis, MO); store at -20°C Osmium tetroxide solution: Dissolve contents of a 0.5-g ampoule of osmium tetroxide (Johnson Matthey, Herts, England or Aldrich, Milwaukee, WI) in 12.5 ml distilled H20 in a glass container (not plastic). Stand at room temperature 2-3 days until dissolved. Store well sealed at 4° for up to 3 months. The solution is diluted (1 part in 5 parts)just before use. Should be used in a fume hood
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HOT stop: 0.3 M sodium acetate (pH 5.2), 0.1 mM EDTA, 25/zg/ml tRNA; store at - 2 0 ° Piperidine: Store at room temperature; 10 M stock (Fluka, Ronkonkoma, NY) is diluted (1 part in 10 parts) before use. Should be used in a fume hood Formamide dye: 90% formamide, 20 mM EDTA, 0.3 mg/ml xylene cyanol, 0.3 mg/ml bromphenol blue; store at - 2 0 ° Acryl/bisacrylamide denaturing gels (8%, w/v): 21 g urea, 10 ml 5 × TBE buffer, 40% acrylamide, distilled H20 to 50 ml. Add 250/zl 10% (w/v) ammonium persulfate and 100/zl N,N,N',N'-tetramethylethylenediamine (TEMED) to set the gel TBE buffer (5 × ): 0.45 M Tris, 0.45 M boric acid, 40 mM EDTA; adjust pH to 8.3 with NaOH Acrylamide (40%, w/v): Make 38 g acrylamide and 2 g N,N'-methylenebisacrylamide up to 100 ml in distilled H20. Deionize by mixing with Bio-Rad (Richmond, CA) analytical-grade mixed bed resin AG501X8(D) 20-50 mesh Bind silane solution: Bring 1 liter distilled H20 to pH 3.5 with acetic acid; add 4 ml Bind Silane (LKB, Rockville, MD) and stir for 15 min Silver staining fixing solution: 10% (v/v) ethanol, 5% (v/v) acetic acid; store at room temperature Silver staining solution: 0.011 mol/liter AgNO3 in distilled H20; make fresh Silver staining developer: 0.75 tool/liter NaOH, 0.1 mol/liter formaldehyde solution, 0.0023 mol/liter sodium borohydride; make fresh Silver staining stop solution: 5% (v/v) acetic acid; store at room temperature Methods
General Method Gel Purification for Probe Preparation. Probes may be gel purified from agarose by dialysis 13 or from a 4% (w/v) nondenaturing acrylamide gel, extracting the DNA by soaking the gel slice in 0.6 M sodium acetate overnight. This allows probe fragments to be separated from contaminants, such as PCR artifacts from PCR-amplified probes, other digestion products from probes made by digestion of larger pieces of DNA, and unincorporated nucleotides from labeled probes. 13 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
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DNA-DNA Duplex Formation. Mix test and control DNAs in equal proportions in 1 x annealing buffer to a final volume from 100 to 400/xl for heteroduplex samples. Homoduplex samples are made using one DNA sample only. Cap the tubes with punctured lids. Incubate in a boiling water bath for 5 min, then quench on ice. Briefly centrifuge the tubes to return water drops to the sample volume. Replace the pierced caps with complete caps and incubate at 42 ° for 1 hr. Place the samples on ice. Ethanol precipitate with 0.3 M sodium acetate (pH 5.3) and 70% (v/v) ethanol. Precipitate the DNA at - 20 ° for 30 min, then centrifuge in a microcentrifuge 13,000 rpm at 4 ° for 15-30 min. Wash the samples with ice-cold 70% (v/v) ethanol. Remove all ethanol drops and air dry the pellets for a few minutes. Resuspend the pellets in distilled H=O. Duplex formation is not necessary if the DNA is PCR amplified because heteroduplex molecules will form during the PCR reaction. 14When heteroduplexes are required between a test and a control DNA, the PCR amplification is performed with both DNA samples in one reaction. Hydroxylamine Modification. Mix 6/xl of duplex with 20/zl hydroxylamine solution. It may be necessary to dissolve the crystals in the hydroxylamine solution at 37 ° before adding it to the DNA samples to begin the reaction. Incubate the samples at 37° for up to 3 hr. (Typical time points are 0 min, 30 min, 1 hr, 2 hr, and 3 hr.) The reaction is stopped by the addition of 200/xl HOT stop and 750/zl ethanol. Precipitate the DNA at - 2 0 ° for 30 rain, spin 13,000 rpm at 4° 15-30 min, and wash pellets in icecold 70% (v/v) ethanol. Air dry pellets for a minute. A time course may be performed in pilot work to identify the best reaction conditions. A zero time point should be included by adding HOT stop before adding the hydroxylamine solution. Homozygous control samples should be included in chemical cleavage experiments. Any banding pattern produced can be assigned as background when these control lanes are compared with heteroduplex samples in which mutations are detected. Osmium Tetroxide Modification. Add 2.5 /xl 10 × osmium tetroxide buffer to 6/zl of duplex samples. Add 15 /xl of freshly diluted osmium tetroxide solution to start the reaction. Mix gently with a pipette tip. A yellow precipitate may form. Incubate samples for up to 5 min at 37°. (Typical time points are 0, 1, and 5 min but may be up to 60 rain in some cases.) Stop the reaction with HOT stop and 70% (v/v) ethanol and precipitate the DNA as described for hydroxylamine reactions. Piperidine Cleavage. Resuspend dry pellets in 50/zl of freshly diluted piperidine. Incubate the samples at 90 ° for 30 rain. Transfer the tubes to 14 C. M. N a g a m i n e , K. C h a n , and Y. F. Lau, A m . J. Hum. Genet. 45, 337 (1989).
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ice. Add 50/zl 0.6 M sodium acetate (pH 5.2), 40/zg glycogen, and 300/zl ethanol. Precipitate the DNA as described for hydroxylamine reactions. Resuspend the samples in 5/zl distilled HzO. Add 2/~1 formamide dye. Electrophoresis. Denature the samples at 95 ° for a few minutes or until the sample volume has decreased to 3-4/zl. Quench the tubes on ice. Run the samples alongside size markers on denaturing acrylamide gels [4-8% (w/v) acrylamide] prewarmed to approximately 50 °. After electrophoresis, dry the gels on Whatman (Clifton, N J) 3MM paper. Autoradiography. Dried gels are placed directly against X-ray film. Initial exposures of 10-16 hr are performed, followed by longer exposures as necessary.
Variations of Method
32p Mode. End labeled or internally radiolabeled [32p]dNTP probes may be used in CCM (refs. 3 and 7, respectively); 6000 disintegrations per minute (dpm) of probe is used in each hydroxylamine or osmium tetroxide reaction. Probes up to 2 kb have been used (Fig. 2). 35S Mode. 35S-Labeled dATP probes may also be used in CCM. In this case 50,000 cpm is used per hydroxylamine or osmium tetroxide reaction. 35S-Labeled probes can give better resolution of larger cleavage products. J5 Probes of sufficiently high specific activity can be difficult to obtain with some DNA samples. Probes of up to 2 kb have been used. Unlabeled Mode. Unlabeled DNA may be screened by CCM. Cleavage products are detected by silver staining. This mode of CCM has the advantage of not requiring use of radioisotopes, but is less sensitive than radiolabeled methods in the detection of multiple cleavage products. Cleavage products from 50 to 550 bp can be detected and DNA fragments of up to 600 bp may be screened for mutations. Fifty nanograms of test and 50 ng of control DNA are used to form heteroduplexes and 100 ng of DNA is used to form homoduplexes. 16Reactions are carried out as outlined in the general method. Acrylamide gels are adhered to gel plates to facilitate staining. Before gels are poured the glass plate is soaked in bind silane solution for 1 hr. The plate is rinsed in clean water and air dried. The gels are then poured as usual. After electrophoresis the gel adhered to a glass plate is briefly rinsed in distilled H20, followed by treatment in fixing solution for 30 min. The gel is rinsed in distilled HzO, then soaked for 2 hr in staining solution. Two brief rinses in distilled H20 follow, then 10-20 min in developer. The gel 15 j. A. Saleeba and R. G. H. Cotton, Nucleic Acids Res. 19, 1712 (1991). 16 S. J. R a m o s and R. G. H. Cotton, Hum. Murat. 1, 63 (1992).
[19]
CHEMICAL CLEAVAGE OF MISMATCH METHOD
293
CONTROL MUTANT H O H O 10 60 1 5 10 60 1 5
425 bp 295 bp
t
1
245 bp
I Q
O
-~-- 115 bp
FIG. 2. Autoradiograph showing mutation detection by chemical cleavage using a [32p] dCTP-labeled probe. The control lanes show homoduplex DNA. No cleavage products are seen after reaction with hydroxylamine for 10 or 60 min, or with osmium tetroxide for I or 5 min. Cleavage products are seen with mutant heteroduplex DNA. Hydroxylamine (H) reaction gives products of 295 and 245 bp. Osmium tetroxide (O) reaction gives products of 425, 295, 245, and 115 bp. (Autoradiograph courtesy of S. M. Forrest of the Murdoch Institute.)
is then stored in stop solution. All incubations are performed with gentle rocking. DNA-RNA Duplexes. A useful adaption of CCM allows DNA-RNA duplexes to be screened for mutation sites.17 In this case 5-10/zg of total RNA or 1 /zg poly(A) RNA is annealed with 5 ng probe DNA. Duplexes are formed by mixing DNA and RNA in 1 x formamide annealing buffer, i7 H.-H. M. Dahl, S. R. Lamande, R. G. H. Cotton, and J. F. Bateman, Anal. Biochem. 183, 263 (1989).
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incubating at 80 ° for 5 min, then at 48 ° for 1 hr. Samples are then precipitated and reactions are performed as described in the general method. Hybond Method. Gogos et al. 18have published an adaptation of CCM whereby probe DNA is hybridized to DNA applied to a membrane. This allows time-consuming ethanol precipitation steps to be simplified. DNA is transferred from one reaction to the next by lifting the membrane between tubes.
Concluding Remarks and Discussion of Problems The CCM method is based on chemical and not enzymatic reactions, and therefore the problems often encountered in molecular biology, for example enzyme reactions that are sensitive to contaminants such as those present in some DNA preparations, do not occur here. However, care must be taken with CCM in correct solution preparation. Overreaction by piperidine is sometimes seen in chemical cleavage. This is indicated by the presence of a ladder of cleavage products at each base position. Repeat the reaction with reduced piperidine reaction time, and check that the piperidine stock is diluted 1 in 10 before use. Lack of mutation detection may be due to a failure to form heteroduplex molecules. Heteroduplexes may be destroyed after formation by heating samples to dissolve DNA pellets. Alternatively, a lack of reaction may be due to aging chemical solutions. These should be remade. Lack of hydroxylamine and osmium tetroxide modification of mismatches is identified by failure of the probe to decrease in intensity with increased incubation time in the presence of these chemicals. In the case of diploid organisms, heteroduplex molecules will be formed if selfoDNA is heated and annealed, when a heterozygous mutation is present. The mutation can then be identified by CCM. If a homozygous mutation is present, CCM with self-DNA will not identify the mutation. In this way heterozygous and homozygous mutations can be distinguished. ~9 Screening more than 2-kb stretches of DNA for mutations may require that a series of probes be designed. These should be arranged so that each probe will overlap neighboring probes by 20-30 bp. Mutations at the ends of probes may be missed by CCM because breathing of the duplex can occur at fragment ends. H Probes obtained by PCR amplification will also Is j. A. Gogos, M. Karayiorgou, H. Aburatani, and F. C. Kafatos, Nucleic Acids Res. 18, 6807 (1990). ~9 I. Dianzani, S. M. Forrest, C. Camaschella, E. Gottardi, and R. G. H. Cotton, Am. J. Hum. Genet. 48, 423 (1991).
[20]
RAPID INTRODUCTION OF RESTRICTION SITES
295
lead to mutations being missed at the ends of probe fragments because regions covered by primers will have primer sequence and not the sequence of mutations, if they were originally present at those sites before amplification. In conclusion, chemical cleavage offers a reliable screening method for mutation detection.
[20] I n t r o d u c i n g R e s t r i c t i o n Sites i n t o D o u b l e - S t r a n d e d Plasmid DNA
By
D A V I D C . K A S L O W a n d DAVID J. RAWLINGS
General Introduction Double-stranded DNA plasmids are the workhorses of molecular biology, especially for cloning foreign genes and expressing recombinant protein. Although a number of pBR322/pUC-derived plasmids now contain a large choice of restriction enzyme sites, many of the specialized vectors for prokaryotic or eukaryotic expression have only a few restriction sites in the cloning area. Oftentimes these sites are incompatible with the fragment to be cloned, or in a case such as ours, a blunt end site and a site for an enzyme that cuts infrequently in the target DNA are not present but are required for high-efficiency blunt end ligation.t The classical approach to introducing new restriction sites is to construct a pair of synthetic oligonucleotides that anneal to one another to yield a double-stranded product that contains single-stranded overhangs at each end complementary to the restriction sites in the vector. This approach is necessary if the restriction site(s) in the vector produce two 5' overhangs, two 3' overhangs, or if one of the sites is blunt ended, if, on the other hand, one of the sites results in a 5' overhang, and the other site has a 3' overhang, then conceivably a single-stranded oligonucleotide could anneal to each end (Fig. 1). This would leave a single-stranded gap in the plasmid that on transformation is filled in by host (bacterial) DNA repair mechanisms. Principle of Method The following is a rapid method for introducing new restriction enzyme sites or any short DNA sequence of choice into virtually any doublei p. Upcroft and A. Healey, Gene 51, 69 (1987).
METHODS IN ENZYMOLOGY,VOL. 217
MUTAGENESIS AND GENE DISRUPTION
[19]
J16, although the latter has a lower AG by 7.5 kcal, there was no difference in the frequency at which mutations were obtained. The methodology for carrying out site-directed mutagenesis has evolved greatly over the past few years. Beyond techniques based on similar methodology as described here, the most significant advance has been in the application of the polymerase chain reaction (PCR) to generate site-directed mutations. A number of protocols have been developed using either two or three oligonucleotide primers. Due to the practical limits of the PCR using Taq DNA polymerase, only a short region is amplified and then cloned into a recipient vector. One potential problem, which has not been extensively documented, is the known lack of fidelity of these thermostable DNA polymerases, especially Taq DNA polymerase, which may introduce second-site mutations in the target. Acknowledgments The authors acknowledge the contributions of Clara Finch, Jonathan Miller, Parke Flick, and Jennifer Webb for supplying data on the mutation frequencies. This work was supported by a grant from the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, the U.S. Army Research Office, and the National Science Foundation.
[19] C h e m i c a l C l e a v a g e o f M i s m a t c h to D e t e c t M u t a t i o n s By JENNIFER A. SALEEBA and RICHARD G. H. COTTON Introduction The chemical cleavage of mismatch (CCM) method allows mutation sites to be detected in kilobase-length pieces of nucleic acids. J A screening method such as this obviates the need to sequence lengths of DNA to determine mutation sites. The method can be widely applied. It can be used to compare a clone of known or unknown sequence to samples of mutant origin, and for applications such as confirmation of in vitro mutagenesis. It has been
J R. G. H. Cotton, N. R. Rodrigues, and D. R. Campbell, Proc. Natl. Acad. Sci. U.S.A. 85, 4397 (1988).
METHODS IN ENZYMOLOGY.VOL. 217
Copyright© 1993by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[19]
CHEMICAL CLEAVAGE OF MISMATCH METHOD
287
widely applied to diagnosis of human inherited disease 2-7 and to mutation detection in other organisms. 8-1°
Principle of the Chemical Cleavage Method Chemical cleavage is based on the principle that mismatched or unmatched residues are more reactive to modification by the chemicals hydroxylamine and osmium tetroxide than matched bases. This can be used to advantage for detection of point mutations and small insertions and deletions. Heteroduplex molecules can be formed by annealing a control and a test piece of DNA. Sites at which residues are mismatched or unmatched will be modified by the chemical hydroxylamine, in the case of cytosine residues, and osmium tetroxide, in the case of thymine residues. Cleavage of the DNA at the site of the modified residues is achieved by subsequent reaction with piperidine. Cleavage products are resolved by denaturing gel electrophoresis, and detection usually takes place by autoradiography (Fig. I). If necessary the exact change present in a mutant is then determined by sequencing the region identified by CCM. Mismatched or unmatched guanine and adenine residues are detected by virtue of the fact that their complementary cytosine and thymine residues are reactive. Cytosine and thymine residues surrounding mismatch sites may have a limited reactivity with hydroxylamine and osmium tetroxide, due to disruption of the helix.11 Analysis of a number of DNA fragments screened for mutations by chemical cleavage has shown that some T residues in T. G mismatches are resistant to modification by osmium tetroxide, and subsequent piperidine 2 j. F. Bateman, S. R. Lamande, H.-H. M., Dahl, D. Chan, T. Mascara, and W. G. Cole, J. Biol. Chem. 264, 10960 (1989). 3 M. Grompe, C. T. Muzny, and C. T. Caskey, Proc. Natl. Acad. Sci. U.S.A. 86, 5888 (1989). 4 A. J. Montandon, P. M. Green, F. Giannelli, and D. R. Bentley, Nucleic Acids Res, 17, 3347 (1989). 5 D. W. Howells, S. M. Forrest, H.-H. M. Dahl, and R. G. H. Cotton, Am. J. Hum. Genet. 47, 279 (1990). I. Dianzani, S, M. Forrest, C. Camaschella, G. Saglio, A. Ponzone, and R. G. H. Cotton, Am. J. Hum. Genet. 48, 631 (1991). 7 S. M. Forrest, H.-H. M. Dahl, D. W. Howells, I. Dianzani, and R. G. H. Cotton, Am. J. Hum. Genet. 49, 175 (1991). 8 R. G. H. Cotton and P. J. Wright, J. Virol. Methods 26, 67 (1989). 9 M. Han and P. W. Steinberg, Cell 63, 921 (1990). l0 M. Grompe, J. Versalovic, T. Koeuth, and J. R. Lupski, J. Bacteriol. 173, 1268 (1991). tl R. G. H. Cotton and R. D. Campbell, Nucleic Acids Res. 17, 4223 (1989).
288
MUTAGENESIS AND GENE DISRUPTION
--T --A
G C
--G ----C
[19]
C G,
I formation Heteroduplex -~C
#
G
~A
C#--'~
I Cleavage H
OT
FIG. 1. Principle behind chemical cleavage. Wild-type and mutant DNAs are heated and annealed to give heteroduplex molecules. Mismatched T resides are reactive to osmium tetroxide (*) and mismatched C residues are reactive to hydroxylamine (#). Cleavage of DNA strands occurs at these sites by reaction with piperidine. Cleavage products are resolved by denaturing gel electrophoresis, and detected by autoradiography or silver staining. A probe band in the first lane and cleavage products after reaction with hydroxylamine (H) and osmium tetroxide (0T) are indicated.
cleavage. 12 The sequence context surrounding the mismatch appears to be important in determining whether a particular T. G mismatch will be modified by osmium tetroxide. Fortunately, a heteroduplex containing a T. G mismatch is always accompanied by a complementary heteroduplex containing an A. C mismatch. An unreactive T- G mismatch can therefore be easily detected by the complementary A. C mismatch, which is highly reactive to hydroxylamine. With this approach, a strategy offering complete detection of mutations by chemical cleavage can be achieved. When using radiolabeled detection, probes are made from both test and control DNAs and used either separately or together in CCM reactions. If using 12 R. G. H. Cotton, manuscript in preparation.
[19]
CHEMICAL CLEAVAGE OF M1SMATCH METHOD
289
a nonradioactive mode, where all DNA is detected, one set of CCM reactions will allow all mutations to be identified. Internally and end-radiolabeled probes and unlabeled DNA may be used to detect cleavage products in CCM. End-labeled probes will be cleaved to reveal one detectable cleavage product for each mutation. This type of probe is best applied to examples in which a number of mutations are expected. If the exact position of the mutation must be known, a single strand should be labeled. Internally radiolabeled probes will give two cleavage products per mutation. These probes are easy to prepare and are therefore well suited to mutation detection in a number of samples in which few or no mutations are expected. Radiolabeled probes made from control and test DNAs must be included either together in one experiment, or separately in a series of experiments, to ensure detection of any unreactive T. G mismatches by their complementary A. C mismatch and completely screen a region of DNA for mutations. Unlabeled DNA may also be used, in which case cleavage products can be detected by methods such as silver staining. All DNA is identified in experiments performed in this way, and therefore complete mutation screening is assured. A variety of nucleic acid templates may be used in chemical cleavage. Cloned fragments, polymerase chain reaction (PCR)-amplified fragments, cDNA, and RNA have all been successfully applied to the identification of mutations.
Materials and Reagents Annealing buffer (2 x ): 1.2 M NaCI, 12 mM Tris-HCl (pH 7.5), 14 mM MgCi2; store at room temperature Formamide annealing buffer: 80% formamide, 40 mM piperazine-N, N'bis(2-ethanesulfonic acid) (PIPES) (pH 6.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.4 M NaC1; store at - 2 0 ° Hydroxylamine solution: Dissolve 1.39 g hydroxylamine hydrochloride or hydroxylammonium chloride (BDH, Poole, England) in 1.6 ml warm distilled HEO in glass. Add 1.75 ml diethylamine dropwise. Final pH should be 6.0. Store at 4° for up to 2 months Osmium tetroxide buffer (10x): 100 mM Tris-HCl (pH 7.7), I0 mM EDTA, 15% (v/v) pyridine (Sigma, St. Louis, MO); store at -20°C Osmium tetroxide solution: Dissolve contents of a 0.5-g ampoule of osmium tetroxide (Johnson Matthey, Herts, England or Aldrich, Milwaukee, WI) in 12.5 ml distilled H20 in a glass container (not plastic). Stand at room temperature 2-3 days until dissolved. Store well sealed at 4° for up to 3 months. The solution is diluted (1 part in 5 parts)just before use. Should be used in a fume hood
290
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HOT stop: 0.3 M sodium acetate (pH 5.2), 0.1 mM EDTA, 25/zg/ml tRNA; store at - 2 0 ° Piperidine: Store at room temperature; 10 M stock (Fluka, Ronkonkoma, NY) is diluted (1 part in 10 parts) before use. Should be used in a fume hood Formamide dye: 90% formamide, 20 mM EDTA, 0.3 mg/ml xylene cyanol, 0.3 mg/ml bromphenol blue; store at - 2 0 ° Acryl/bisacrylamide denaturing gels (8%, w/v): 21 g urea, 10 ml 5 × TBE buffer, 40% acrylamide, distilled H20 to 50 ml. Add 250/zl 10% (w/v) ammonium persulfate and 100/zl N,N,N',N'-tetramethylethylenediamine (TEMED) to set the gel TBE buffer (5 × ): 0.45 M Tris, 0.45 M boric acid, 40 mM EDTA; adjust pH to 8.3 with NaOH Acrylamide (40%, w/v): Make 38 g acrylamide and 2 g N,N'-methylenebisacrylamide up to 100 ml in distilled H20. Deionize by mixing with Bio-Rad (Richmond, CA) analytical-grade mixed bed resin AG501X8(D) 20-50 mesh Bind silane solution: Bring 1 liter distilled H20 to pH 3.5 with acetic acid; add 4 ml Bind Silane (LKB, Rockville, MD) and stir for 15 min Silver staining fixing solution: 10% (v/v) ethanol, 5% (v/v) acetic acid; store at room temperature Silver staining solution: 0.011 mol/liter AgNO3 in distilled H20; make fresh Silver staining developer: 0.75 tool/liter NaOH, 0.1 mol/liter formaldehyde solution, 0.0023 mol/liter sodium borohydride; make fresh Silver staining stop solution: 5% (v/v) acetic acid; store at room temperature Methods
General Method Gel Purification for Probe Preparation. Probes may be gel purified from agarose by dialysis 13 or from a 4% (w/v) nondenaturing acrylamide gel, extracting the DNA by soaking the gel slice in 0.6 M sodium acetate overnight. This allows probe fragments to be separated from contaminants, such as PCR artifacts from PCR-amplified probes, other digestion products from probes made by digestion of larger pieces of DNA, and unincorporated nucleotides from labeled probes. 13 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
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DNA-DNA Duplex Formation. Mix test and control DNAs in equal proportions in 1 x annealing buffer to a final volume from 100 to 400/xl for heteroduplex samples. Homoduplex samples are made using one DNA sample only. Cap the tubes with punctured lids. Incubate in a boiling water bath for 5 min, then quench on ice. Briefly centrifuge the tubes to return water drops to the sample volume. Replace the pierced caps with complete caps and incubate at 42 ° for 1 hr. Place the samples on ice. Ethanol precipitate with 0.3 M sodium acetate (pH 5.3) and 70% (v/v) ethanol. Precipitate the DNA at - 20 ° for 30 min, then centrifuge in a microcentrifuge 13,000 rpm at 4 ° for 15-30 min. Wash the samples with ice-cold 70% (v/v) ethanol. Remove all ethanol drops and air dry the pellets for a few minutes. Resuspend the pellets in distilled H=O. Duplex formation is not necessary if the DNA is PCR amplified because heteroduplex molecules will form during the PCR reaction. 14When heteroduplexes are required between a test and a control DNA, the PCR amplification is performed with both DNA samples in one reaction. Hydroxylamine Modification. Mix 6/xl of duplex with 20/zl hydroxylamine solution. It may be necessary to dissolve the crystals in the hydroxylamine solution at 37 ° before adding it to the DNA samples to begin the reaction. Incubate the samples at 37° for up to 3 hr. (Typical time points are 0 min, 30 min, 1 hr, 2 hr, and 3 hr.) The reaction is stopped by the addition of 200/xl HOT stop and 750/zl ethanol. Precipitate the DNA at - 2 0 ° for 30 rain, spin 13,000 rpm at 4° 15-30 min, and wash pellets in icecold 70% (v/v) ethanol. Air dry pellets for a minute. A time course may be performed in pilot work to identify the best reaction conditions. A zero time point should be included by adding HOT stop before adding the hydroxylamine solution. Homozygous control samples should be included in chemical cleavage experiments. Any banding pattern produced can be assigned as background when these control lanes are compared with heteroduplex samples in which mutations are detected. Osmium Tetroxide Modification. Add 2.5 /xl 10 × osmium tetroxide buffer to 6/zl of duplex samples. Add 15 /xl of freshly diluted osmium tetroxide solution to start the reaction. Mix gently with a pipette tip. A yellow precipitate may form. Incubate samples for up to 5 min at 37°. (Typical time points are 0, 1, and 5 min but may be up to 60 rain in some cases.) Stop the reaction with HOT stop and 70% (v/v) ethanol and precipitate the DNA as described for hydroxylamine reactions. Piperidine Cleavage. Resuspend dry pellets in 50/zl of freshly diluted piperidine. Incubate the samples at 90 ° for 30 rain. Transfer the tubes to 14 C. M. N a g a m i n e , K. C h a n , and Y. F. Lau, A m . J. Hum. Genet. 45, 337 (1989).
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ice. Add 50/zl 0.6 M sodium acetate (pH 5.2), 40/zg glycogen, and 300/zl ethanol. Precipitate the DNA as described for hydroxylamine reactions. Resuspend the samples in 5/zl distilled HzO. Add 2/~1 formamide dye. Electrophoresis. Denature the samples at 95 ° for a few minutes or until the sample volume has decreased to 3-4/zl. Quench the tubes on ice. Run the samples alongside size markers on denaturing acrylamide gels [4-8% (w/v) acrylamide] prewarmed to approximately 50 °. After electrophoresis, dry the gels on Whatman (Clifton, N J) 3MM paper. Autoradiography. Dried gels are placed directly against X-ray film. Initial exposures of 10-16 hr are performed, followed by longer exposures as necessary.
Variations of Method
32p Mode. End labeled or internally radiolabeled [32p]dNTP probes may be used in CCM (refs. 3 and 7, respectively); 6000 disintegrations per minute (dpm) of probe is used in each hydroxylamine or osmium tetroxide reaction. Probes up to 2 kb have been used (Fig. 2). 35S Mode. 35S-Labeled dATP probes may also be used in CCM. In this case 50,000 cpm is used per hydroxylamine or osmium tetroxide reaction. 35S-Labeled probes can give better resolution of larger cleavage products. J5 Probes of sufficiently high specific activity can be difficult to obtain with some DNA samples. Probes of up to 2 kb have been used. Unlabeled Mode. Unlabeled DNA may be screened by CCM. Cleavage products are detected by silver staining. This mode of CCM has the advantage of not requiring use of radioisotopes, but is less sensitive than radiolabeled methods in the detection of multiple cleavage products. Cleavage products from 50 to 550 bp can be detected and DNA fragments of up to 600 bp may be screened for mutations. Fifty nanograms of test and 50 ng of control DNA are used to form heteroduplexes and 100 ng of DNA is used to form homoduplexes. 16Reactions are carried out as outlined in the general method. Acrylamide gels are adhered to gel plates to facilitate staining. Before gels are poured the glass plate is soaked in bind silane solution for 1 hr. The plate is rinsed in clean water and air dried. The gels are then poured as usual. After electrophoresis the gel adhered to a glass plate is briefly rinsed in distilled H20, followed by treatment in fixing solution for 30 min. The gel is rinsed in distilled HzO, then soaked for 2 hr in staining solution. Two brief rinses in distilled H20 follow, then 10-20 min in developer. The gel 15 j. A. Saleeba and R. G. H. Cotton, Nucleic Acids Res. 19, 1712 (1991). 16 S. J. R a m o s and R. G. H. Cotton, Hum. Murat. 1, 63 (1992).
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CONTROL MUTANT H O H O 10 60 1 5 10 60 1 5
425 bp 295 bp
t
1
245 bp
I Q
O
-~-- 115 bp
FIG. 2. Autoradiograph showing mutation detection by chemical cleavage using a [32p] dCTP-labeled probe. The control lanes show homoduplex DNA. No cleavage products are seen after reaction with hydroxylamine for 10 or 60 min, or with osmium tetroxide for I or 5 min. Cleavage products are seen with mutant heteroduplex DNA. Hydroxylamine (H) reaction gives products of 295 and 245 bp. Osmium tetroxide (O) reaction gives products of 425, 295, 245, and 115 bp. (Autoradiograph courtesy of S. M. Forrest of the Murdoch Institute.)
is then stored in stop solution. All incubations are performed with gentle rocking. DNA-RNA Duplexes. A useful adaption of CCM allows DNA-RNA duplexes to be screened for mutation sites.17 In this case 5-10/zg of total RNA or 1 /zg poly(A) RNA is annealed with 5 ng probe DNA. Duplexes are formed by mixing DNA and RNA in 1 x formamide annealing buffer, i7 H.-H. M. Dahl, S. R. Lamande, R. G. H. Cotton, and J. F. Bateman, Anal. Biochem. 183, 263 (1989).
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incubating at 80 ° for 5 min, then at 48 ° for 1 hr. Samples are then precipitated and reactions are performed as described in the general method. Hybond Method. Gogos et al. 18have published an adaptation of CCM whereby probe DNA is hybridized to DNA applied to a membrane. This allows time-consuming ethanol precipitation steps to be simplified. DNA is transferred from one reaction to the next by lifting the membrane between tubes.
Concluding Remarks and Discussion of Problems The CCM method is based on chemical and not enzymatic reactions, and therefore the problems often encountered in molecular biology, for example enzyme reactions that are sensitive to contaminants such as those present in some DNA preparations, do not occur here. However, care must be taken with CCM in correct solution preparation. Overreaction by piperidine is sometimes seen in chemical cleavage. This is indicated by the presence of a ladder of cleavage products at each base position. Repeat the reaction with reduced piperidine reaction time, and check that the piperidine stock is diluted 1 in 10 before use. Lack of mutation detection may be due to a failure to form heteroduplex molecules. Heteroduplexes may be destroyed after formation by heating samples to dissolve DNA pellets. Alternatively, a lack of reaction may be due to aging chemical solutions. These should be remade. Lack of hydroxylamine and osmium tetroxide modification of mismatches is identified by failure of the probe to decrease in intensity with increased incubation time in the presence of these chemicals. In the case of diploid organisms, heteroduplex molecules will be formed if selfoDNA is heated and annealed, when a heterozygous mutation is present. The mutation can then be identified by CCM. If a homozygous mutation is present, CCM with self-DNA will not identify the mutation. In this way heterozygous and homozygous mutations can be distinguished. ~9 Screening more than 2-kb stretches of DNA for mutations may require that a series of probes be designed. These should be arranged so that each probe will overlap neighboring probes by 20-30 bp. Mutations at the ends of probes may be missed by CCM because breathing of the duplex can occur at fragment ends. H Probes obtained by PCR amplification will also Is j. A. Gogos, M. Karayiorgou, H. Aburatani, and F. C. Kafatos, Nucleic Acids Res. 18, 6807 (1990). ~9 I. Dianzani, S. M. Forrest, C. Camaschella, E. Gottardi, and R. G. H. Cotton, Am. J. Hum. Genet. 48, 423 (1991).
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lead to mutations being missed at the ends of probe fragments because regions covered by primers will have primer sequence and not the sequence of mutations, if they were originally present at those sites before amplification. In conclusion, chemical cleavage offers a reliable screening method for mutation detection.
[20] I n t r o d u c i n g R e s t r i c t i o n Sites i n t o D o u b l e - S t r a n d e d Plasmid DNA
By
D A V I D C . K A S L O W a n d DAVID J. RAWLINGS
General Introduction Double-stranded DNA plasmids are the workhorses of molecular biology, especially for cloning foreign genes and expressing recombinant protein. Although a number of pBR322/pUC-derived plasmids now contain a large choice of restriction enzyme sites, many of the specialized vectors for prokaryotic or eukaryotic expression have only a few restriction sites in the cloning area. Oftentimes these sites are incompatible with the fragment to be cloned, or in a case such as ours, a blunt end site and a site for an enzyme that cuts infrequently in the target DNA are not present but are required for high-efficiency blunt end ligation.t The classical approach to introducing new restriction sites is to construct a pair of synthetic oligonucleotides that anneal to one another to yield a double-stranded product that contains single-stranded overhangs at each end complementary to the restriction sites in the vector. This approach is necessary if the restriction site(s) in the vector produce two 5' overhangs, two 3' overhangs, or if one of the sites is blunt ended, if, on the other hand, one of the sites results in a 5' overhang, and the other site has a 3' overhang, then conceivably a single-stranded oligonucleotide could anneal to each end (Fig. 1). This would leave a single-stranded gap in the plasmid that on transformation is filled in by host (bacterial) DNA repair mechanisms. Principle of Method The following is a rapid method for introducing new restriction enzyme sites or any short DNA sequence of choice into virtually any doublei p. Upcroft and A. Healey, Gene 51, 69 (1987).
METHODS IN ENZYMOLOGY,VOL. 217