HUMAN MUTATION 1:63-69 (1992)

METHODS

Complete Mutation Detection using Unlabeled Chemical Cleavage Jennifer A. Saleeba, Susan J. Ramus, and Richard G.H. Cotton' Olive Miller Protein Laboratory, Murdoch Institute, Royal Children's Hospital, Fkmington Road, ParkviUe, Victoria 3052. Awtrdia Communicated by Darwin I. Prockop

We have developed a strategy for the complete detection of point mutations, small insertions and deletions by chemical cleavage based on the methodology of Cotton et al. (1988). The technique was extended by the development of a nonisotopic cleavage product detection system using silver staining after gel electrophoresis.The complete mutation detection w a s achieved by use of mutant and wild-type DNAs in equimolar quantities in duplex formation,thus any mismatches that are resistant to chemical cleavage (e.g., some T-G mismatches) are easily detected by cleavage of the complementary heteroduplex (e.g., A-C mismatch). With such a strategy mutant DNAs can be screened for mutations and polymorphisms. The advantages of complete unlabeled mutation detection are considerable. 8 1992 Wdey-Liss, Inc. KEY WORDS:

P-thalassemia, Sickle cell anemia, Nonradioactive, DNA mutations, Silver stain

INTRODUCTION Detection of mutations and polymorphisms in DNA is an important feature of the investigation of gene structure and function. Furthermore, easy identification of specific sequences and sequence changes plays a central role in the diagnosis of human inherited diseases. It is not surprising, therefore, that there has been considerable interest in the field of mutation detection (Cotton, 1989; Rossiter and Caskey, 1990; Cotton, 1991, for recent reviews). A number of approaches have been taken to scanning methods of mutation detection whereby hundreds of base pairs are scanned for mutations in a single experiment. One method is based on the principle that denatured DNA samples of different sequence have small differences in their electrophoretic mobilities (Orita et al., 1989a,b). Similarly, electrophoresis of DNA through a gradient of denaturant may give different electrophoretic mobility for heteroduplex molecules compared to that of homoduplex molecules (Myers et al., 1985a). These methods can scan fragments of a few hundred base pairs of DNA for the presence of most mutations. Ganguly et al. (1989) have demonstrated that carbodiimide complexed with mismatched base pairs in heteroduplexes will stall the progression of DNA polymerase in a primer exten0

1992 WILEY-LISS,INC.

sion reaction, thereby allowing the mutation sites to be located. RNase A cleavage of mismatch sites in RNA:DNA or RNA:RNA heteroduplexes was developed by Myers et al. (1985b); however, not all classes of mismatches are reactive. In the chemical cleavage method (CCM) heteroduplex molecules are formed between test and control DNA samples. Hydroxylamine and osmium tetroxide react to modify mismatched or unmatched cytosine or thymine residues, respectively. Sites modified by hydroxylamine and osmium tetroxide are more susceptible to cleavage by piperidine than unmodified base pairs. Piperidine is therefore used to cleave DNA fragments at the site of modified bases. Cleavage occurs in one strand of the helix only. Products are resolved by denaturing electrophoresis to allow the identification and location of mutation sites. Chemical cleavage was shown to be an excellent method for the detection and location of mutations, particularly because it is possible to easily scan up to 2 kb segments of DNA at a time and DNA or RNA

Received December 11, 1991; accepted Janualy 9, 1992 *Towhom reprint requestdcorrespondence should be addressed. Abbreviations: A, adenine; G, guanine; C, cytosine; T, thymine; &TP, adenine triphosphate; dCTP, cytosine triphosphate; kb, kilobase pairs.

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templates may be used (Cotton et al., 1988; Dahl et al., 1989). Improved resolution of larger cleavage products is possible by the use of "S-labeled dATP probes rather than [32P]dCTP-labeled probes (Saleeba and Cotton, 1991). Mutation detection by chemical cleavage has been applied to the analysis of gene structure in human systems and in other organisms such as Caenorhabditis elegans (Han and Sternberg, 1990), Escherichia coli (Grompe et al., 1991), and dengue virus (Cotton and Wright, 1989). The human diseases investigated by the method include P-thalassemia (Dianzani et al., 1991a), osteogenesis imperfecta (Bateman et al., 1989; Lamande et al., 1989), dihydropteridine reductase deficiency (Howells et al., 1990), pyruvate dehydrogenase deficiency (Dahl et al., 1990), colorectal cancer (Rodrigues et al., 1990), ornithine transcarbamylase deficiency (Grompe et al., 1989), and phenylketonuria (Forrest et al., 1991). Analysis of the growing literature using the chemical cleavage method showed that some mismatches appear to be resistant to detection by CCM (Forrest et al., 1991; R.G.H.C., unpublished data). We present here a strategy that overcomes this limitation and allows complete mutation detection by chemical cleavage. An unlabeled system is used that is capable of detecting mutations including those resistant to chemical cleavage in one sense. MATERIALS AND METHODS Genomic DNA

Human DNA samples were prepared from blood (Miller et al., 1988). DNA Amplification and Purification

A 627-bp segment of the human p-globin gene was PCR amplified using the method and primers a and b described by Dianzani et al. (1991a). Products were purified by electroelution (Sambrook et al., 1989). Duplex Formation

Test and control DNA (50 ng each) were mixed in annealing buffer (Cotton et al., 1988) for heteroduplex formation. DNA (100 ng) from one source was used for homoduplex formation. This amount of DNA is sufficient for resolution of the products of one chemical cleavage reaction in one gel track. Quantities were increased according to the number of samples required. Samples were placed in a boiling water bath for 5 min, transferred to ice, then incubated at 42°C for 1 hr.

After annealing, samples were transferred to ice and ethanol precipitated. Chemical Cleavage and Gel Electrophoresis

Chemical cleavage and the resolution of cleavage products were performed as described (Howells et al., 1990), but with the following modifications. Glycogen carrier (40 pg) was added to samples after reaction with piperidine, and samples were ethanol precipitated at -20°C for 30 min. After piperidine reaction samples were ethanol precipitated, then resuspended in 5 pl dH20. Formamide dye (2 pl) was then added. Samples were incubated at 95°C for 5 min to denature the DNA and to reduce sample volume by evaporation to 2-4 pl. Samples were quenched on ice before loading onto prerun denaturing polyacrylamide gels prepared in the following manner. One glass plate from the gel electrophoresis apparatus was soaked for 1 hr in Bind Silane (Pharmacia LKB Biotechnology, Uppsala, Sweden) prepared by the manufacture's instructions, briefly rinsed in dH20, and allowed to air dry before the gel apparatus was assembled and the acrylamide gel was poured. This procedure was used to adhere the acrylamide gels to the gel plate to facilitate staining. Detection by Silver Staining

Denaturing polyacrylamide gels (400 X 250 x 0.4 mm) were silver stained as described (Leibermann et al., 1990). The stain was developed for 20 min. Permanent record of results were obtained by photography with back illumination. Some decrease in the clarity of cleavage bands occurred with photography therefore original gels, as well as photographs, were examined for results. Gels may be stored by air drying between cellophane sheets. RESULTS Detection of Mutations in the Gene Encoding Human P-Globin Causing P-Thalassemia

Figure l a shows detection of the -87 (C + G) mutation (Orkin et al., 1982) in the gene encoding human P-globin. A 627-bp homoduplex made with wildtype DNA shows no cleavage products. A heteroduplex formed between mutant and wildtype DNAs shows cleavage of the PCR fragment revealing the expected 90-bp fragment. No cleavage, however, is observed in mutant duplex DNA, indicating that the mutation is homozygous. Figure l b shows detection of the codon 6 deletion mutation (Kazazian et al., 1983) in the gene encoding human P-globin. Duplexes formed be-

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FIGURE1. (a) Detection by CCM of an individual homozygous for the -87 mutation in human p-globin causing p-thalassemia. Wild-type homoduplex DNA was treated for 0 and 3 hr with hydroxylamine (lanes 6 and 5, respectively). No cleavage products are seen. Wild-typelmutant duplex DNA was subjected to the same reaction conditions (lanes 4 and 3, respectively). The 627-bp probe from the 5' region of pglobin was cleaved revealing a 90-bp fragment after 3-hr hydroxylamine treatment. The 537-bp cleavage product also produced is not resolved on this gel. The 90-bp product is not seen with hydroxylamine treatment of mutant duplex DNA indicating that the mutation is homozygous (lane 2 shows 0-hr treatment and lane 1 shows 3-hr treatment). DNA was visualized by silver staining. (b) Detection by CCM of an

individual heterozygous for the codon 6 mutation in human p-globin causing p-thalassemia. Wild-type homoduplex DNA is shown after treatment for 0 (lane 1)and 5 min (lane 2) with osmium tetroxide. No cleavage products are seen. Wild-Wmutant duplex DNA samples are shown after reaction under the same reaction conditions (lane 3 shows 0min reaction and lane 4 shows 5-min reaction). The 627-bp probe from the 5' region of p-globin was cleaved revealing 386-bp and 241-bp fragments after 5-min osmium tetroxide treatment. The cleavage products are also seen with osmium tetroxide treatment of mutant duplex sample (0-min reaction in lane 5, and 5-min reaction in lane 6) indicating that the mutation is heterozygous. DNA was visualised by silver staining.

tween wild-type and mutant DNAs and self-mutant duplexes are cleaved indicating that the mutant DNA sample is heterozygous for the codon 6 deletion mutation. Cleavage products are more easily identified in self-mutant duplex samples because a higher proportion of DNA will form heteroduplex molecules and be cleaved, giving a higher signal to background than in duplexes formed between wild-type and mutant DNAs. Further examples of mutation detection in human P-globin are shown in Figure 2. A 627-bp region of the gene was screened for mutations in 3 DNA samples. One sample was homozygous for the nonsense codon 39 (C --j T) mutation (Orkin and Goff, 1981; Moschonas et al., 1981; Trecartin et al., 1981), another is a compound heterozygote for the codon 39 and IVS-1 position 110 (G + A) (Spritz et al., 1981; Westaway and Williamson, 1981) mutations, and a compound heterozygote for the codon 39 and IVS-1 position 1 (G + A) (Orkin et al., 1982) mutations.

Detection of the Sickle Mutation in the Gene Encoding Human P-Globin Figure 3 shows the detection of the A to T transition mutation that gives rise to sickle cell anemia (Marotta et al., 1977). The DNA sample is homozygous for the mutation. Previous attempts at the detection of this mutation using chemical cleavage with [32P]dCTP-labeledwild-type DNA used as the probe gave only a weak signal (unpublished). This is thought to be due to poor cleavage at the site of the mismatched T residue in the wild-type DNA strand. Detection of the mutation was more efficient when the chemical cleavage procedure enabled both wild-type and mutant strands to be assayed. This is presumably because cleavage at the site of the T.T mismatch occurs more efficiently in the mutant strand then in the wild-type strand, due to the sequence context of the mismatch. This example illustrates how the approach to chemical cleavage described here gives thorough mutation screening.

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Detection by CCM of 2 compound heterozygous individuals with mutations in human p-globin giving pthalassernia. Wild-type/mutant duplex DNA is shown for each sample after reaction for 3 hr with hydroxylamine. In both samples the 627-bp probe from the 5’ region of Pglobin was cleaved revealing 474-bp and 153-bp fragments. This identifies the codon 39 mutation in both samples. The sample in track 1 also shows 428-bp and 199-bp cleavage products. This identifies the IVS-1-110 mutation. Track 2 also shows another mutation. The 319-bp and 308-bp cleavage products identify the NS-1-1 mutation. DNA was visualized by silver staining. FIGURE 2.

Detection of Other Mutations

This approach to the detection of mutations by chemical cleavage has also been successfully applied to the detection of the F39L mutation (Forrest et al., 1991) in human phenylalanine hydroxylase that gives rise to phenylketonuria (data not shown). DISCUSSION

A strategy has been developed for the complete detection of point mutations, small insertions, and deletions by unlabeled chemical cleavage. Heteroduplexes are formed between wild-type and mutant DNA samples. Mismatches, unmatched, and nearby residues are susceptible to chemical cleavage (Cotton and Campbell, 1989), and the site of mismatches can be calculated from the pattern of cleavage products. Heterozygous or homozygous mutations can be immediately differentiated by the inclusion of a mutant DNA duplex in reaction samples. If reactive residues are also detected in these samples the mutation must be heterozygous. If no cleavage products are seen the mutation is homozygous (Dianzani et al. , 1991b) . A survey of reactive and nonreactive mis-

FIGURE 3. Detection by CCM of an individual homozygous for the HbS mutation in human p-globin causing sickle cell anemia. Wild-type homoduplex DNA is shown with treatment for zero (lane 1) and 5 min (lane 2) with osmium tetroxide. No cleavage products are seen. Wild-typelmutant duplex DNA was subjected to the same reaction conditions (lanes 3 and 4). The 627-bp probe from the 5’ region of p-globin was cleaved to reveal 381-bp and 246-bp fragments after 5-min osmium tetroxide treatment (lane 4) that were not present with 0 reaction time (lane 3). No products are seen with osmium tetroxide treatment of mutant duplex DNA, indicating that the mutation is homozygous (lane 5 shows reaction for 0 min, and lane 6 shows reaction for 5 min). DNA was visualized by silver staining.

matches has identified a class of mismatches that may be resistant to chemical cleavage (R.G.H.C., unpublished data). This is thought to be due to stacking interactions surrounding a relatively stable mismatch (generally T*G) in a certain sequence context rendering the mismatch less reactive to chemical cleavage than other mismatches. Although there are strong stacking forces surrounding this mismatch, the less stable complementary mismatch (generally A C ) is surrounded by weak stacking forces, which renders this mismatch highly reactive to chemical cleavage. Advantage may be taken of the highly reactive nature of this mismatch in detection of the mutation (Fig. 4). A method which allows all cleavage products from both duplex molecules to be readily detected will ensure complete mutation detection. This means each mutation is represented by two mismatches of different stability, each in a different sequence context. Detection of DNA by silver staining achieves this result. Radiolabeled DNA also allows for complete detection if test and con-

COMPLETE MUTATION DETECTION

Mutant

G C

W il d t y p e

A T

Heteroduplex f o r m a t i o n

1 G T

'* I

A

C# FIGURE4. Strategy for complete chemical cleavage. G*Tmismatches (*) unreactive with osmium tetroxide can easily be detected by hydroxylamine reaction of the complementary A C mismatch (#). If cleavage products from wild-type DNA only is assayed. TOG mismatches unreactive to osmium tetroxide may be missed. Complete detection is achieved if cleavage products are assayed from duplex molecules formed by mixing mutant and wild-type DNAs in equimolar amounts, such as in silver staining.

trol DNAs are used as probes together or in separate experiments (Forrest et al., 1991). Complete detection of mutations is described using a gene scanning method rather than sequencing of every base pair. The applications of the method are widespread. The complete chemical cleavage method (CCCM) may be used for quick confirmation of in vitro mutagenesis, for the identification of new mutations, and for routine screening for mutations such as in the diagnosis of human inherited diseases. We have illustrated the successful use of CCCM here in the identification of 6 human P-globin mutations. Deletion, transition and transversion mutations were identified with examples covering homozygous and heterozygous cases. Cleavage products were identified most clearly

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when a minimum amount of homoduplex sample was present with the reactive heteroduplex molecules. This will occur when mutant and wild-type DNA strands are present in equimolar quantities, either in duplex formation with self-heterozygous DNA, or when mutant and wild-type DNAs are mixed in equal amounts if the mutation is homozygous. A higher signal to background signal is achieved under these conditions. In the diagnosis of human inherited disease, an affected individual often requires the presence of 2 affected alleles. Most mutations applicable to CCM screening could be expected to be identified in a compound heterozygous state. Samples could be initially screened annealed to self DNA. This mode would give the most sensitive detection of mutations. If this test failed to identify causative mutations, the test sample could be mixed with control DNA and CCM performed to identify homozygous mutations. Carrier detection would also be possible in a self DNA CCM screen. The method presented here also has the advantage of not relying on any labeling method. No expensive protective equipment is required, nor are special waste management or staff training procedures needed as are required for handling radiolabeled substances. No potentially rate-limiting labeling step is required and final results are obtained within a few hours of electrophoresis. The silver staining detection method produces very discrete banding patterns and has the potential to be automated (Dockhorn-Dworniczaket al., 1991). Most importantly, the method is available for diagnosis in countries where radiolabeled substances are not readily available. The protocol also requires relatively low technology, requiring PCR capability, an extraction fan, and an acrylamide gel apparatus. Potential disadvantages of the method presented here are that more DNA is required than in the labeled procedure. This is not a serious concern, however, with the wide application of PCR technology allowing amplification of DNA segments to be scanned for mutations. DNA amplified from one standard 100 ~1 PCR reaction is sufficient to screen an individual using this method. A difference between detection of cleavage products by silver staining and autoradiography of labeled cleavage products is that with labeled products a series of exposures may be performed to view larger products, which give a strong signal, and smaller products, which give a weaker signal. A series of detection intensities may be achieved with silver staining by electrophoresis of a series of CCM samples performed on a variety of DNA

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quantities. We have found that a single set of cleavage samples will screen for mutations over a 600-bp region. Best detection was obtained with cleavage products in the range of sizes from 50 to 630 bp, and in cases where fewer than five cleavage products were obtained. Presumably electrophoretic separation of larger cleavage products would allow mutation screening over much larger lengths of DNA. We have presented here an unlabeled approach to complete mutation detection using chemical cleavage. The method can reliably screen 600 bp segments of DNA for mutations. Of the methods for detecting unknown mutations in current use, sequencing and complete chemical cleavage (presented here) have undergone most extensive trials and have the ability to identify all mutations and locate them precisely. Errors can occur in the reading of sequencing gels, but this is not a problem in CCCM where experiments identify only mutation sites. Sequencing has the advantage over CCCM in immediately identifying the exact mutational change in a sample, but this may not be essential information in all applications such as in some prenatal diagnoses. In summary, improvements in the chemical cleavage method of mutation detection have produced a precision method. ACKNOWLEDGMENTS

DNA samples were kindly provided by Drs. 1. Dianzani and C. Camaschella. Drs. S.M. Forrest, P.M. Smooker, and H-H.M. Dahl are gratefully acknowledged for comments on the manuscript. This work was supported by a Victorian Health Promotion Foundation Research Grant. REFERENCES BatemanJF, Lamande SR, Dahl H-HM, Chan D, Mascara T, Cole WG (1989) A frameshift mutation results in a truncated nonfunctional carboxyl-terminalproalphal(1) propeptide of type I collagen in osteogenesis imperfecta. J Biol Chem 264: 1096010964. Cotton RGH (1989) Detection of single base changes in nucleic acids. Biochem J 263:l-10. Cotton RGH (1991) Detection of single base changes in nucleic acid. In Verma R (ed): Advances in Genome Biology. Unfolding the Genome. Greenwich, CT: JAI Press, Vol. 1, pp 253300. Cotton RGH, Campbell RD (1989) Chemical reactivity of matched cytosine and thymine bases near mismatched and unmatched bases in a heteroduplex between DNA strands with multiple differences. Nucleic Acids Res 17:4223-4233. Cotton RGH, Rogrigues NR, Campbell RD (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 85:4397-4401. Cotton RGH, Wright PJ (1989) Rapid chemical mapping of

dengue virus variability using RNA isolated directly from cells. J Virol Methods 26:67-76. Dahl H-HM, Lamamde SR, Cotton RGH, Bateman JF (1989) Detection and localization of base changes in RNA using a chemical cleavage method. Anal Biochem 183:263-268. Dahl H-HM, Maragos C, Brown RM, Hansen L, Brown GK ( 1990) Pyruvate dehydrogenase deficiency caused by deletion of a 7-bp repeat sequence in the Elalpha gene. Am J Hum Genet 47:286-293. Dianzani I, Camaschella C, Saglio G, Forrest SM, Ramus S, Cotton RGH (1991a) Simultaneousscreening for beta-thalassemia mutations by chemical cleavage of mismatch. Genomics 11: 48-53. Dianzani I, Forrest SM, Camaschella C, Gottardi E, Cotton RGH (1991b) Heterozygotes and homozygotes: Discrimination by chemical cleavage of mismatch. Am J Hum Genet 48:423424. Dockhorn-Dworniczak B, Dworniczak B, Brommelkamp L, Bulles 1, Horst J, Bocker WW (1991) Non-isotopic detection of single strand conformation polymorphism (PCR-SSCP): A rapid and sensitive technique in diagnosis of phenylketonuria. Nucleic Acids Res 19:2500. Forrest SM, Dahl H-HM, Howells DW, Dianzani I, Cotton RGH (1991) Mutation detection in phenylketonuria by chemical cleavage of mismatch: Importance of using probes from both normal and patient samples. Am J Hum Genet 49:175-183. Ganguly A, Rooney jE, Hosomi S, Zeiger AR, Prockop DJ (1989) Detection and location of single-basemutations in large DNA fragments by immunomicroscopy. Genomics 4:530-538. Grompe M, Muzny M, Caskey CT (1989) Scanning detection of mutations in human ornithine transcarbamoylaseby chemical mismatch cleavage. Proc Natl Acad Sci USA 86:5888-5892. Grompe M, Versalovic J , Koetuth T, Lupski JR (1991) Mutations in the E s c h c h i a coli dnaC gene suggest coupling between DNA replication and chromosome partitioning. J Bacteriol 173:1268-1278. Han M, Sternberg PW (1990) fet-60, a gene that specifies cell fate during C. ekganr vulva1 induction, encodes a ras protein. Cell 63:921-931. Howells DW, Forrest SM, Dahl H-HM, Cotton RGH (1990) Insertion of an extra codon for threonine is a cause of dihydropteridine reductase deficiency. Am J Hum Genet 47:279-285. Kazazian HH Jr, Orkin SH, Boehm CD, Sexton JP, Antonarakis SE (1983) Beta-Thalassemiadue to a deletion of the nucleotide which is substituted in the beta S-globin gene. Am J Hum Genet 35:1028-1033. Lamande SR, Dahl H-HM, Cole WG, Bateman JF (1989) Characterization of point mutations in the collagen COLtAl and COLlA2 genes causing lethal perinatal osteogenesis imperfecta. J Biol Chem 264:15809-15812. Leibermann HT, Fedtke C, Kwasniowski W (1990) Detection of T1-oligonucleotidesof the foot-and-mouth disease virus DNA by silver staining. Acta Virol 34:202-205. Marotta CA, Wilson JT, Forget BC, Weissman SM (1977) Human beta-globin messenger RNA. I. Nucleotide sequences derived from complementary RNA. J Biol Chem 252:5040-5053. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215. Moschonas N , de Boer E, Grosveld FG, Dahl H-HM, Wright S, Shewmaker CK, Flavell RA (1981) Structure and expression of a cloned beta" thalassaemic globin gene. Nucleic Acids Res 9:4391-4401. Myers RM, Lumelsky N, Lerman LS, Maniatis T (1985a) Detection of single base substitutions in total genomic DNA. Nature (London) 313:495-498.

COMPLETE MUTATION DETECTION Myers RM, Fischer SG, Lerman LS, Maniatis T (1985b) Nearly all single base substitutions in DNA fragments joined to a GCclamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131-3145. Orita M, Suzuki Y, Sekiya T, Hayashi K (1989a) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5874-879. Orita M, lwahana H, Kanazawa H, Hayashi K, Sekiya T (1989b) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766-2770. Orkni SH, Goff SC (1981) Nonsense and kameshift mutations in beta 0-thalassemia detected in cloned beta-globin genes. J Biol Chem 256:9782-9784. Orkin SH, Kazazian HHJr, Antonarakis SE, Goff SC, Boehm CD, Sexton JP, Waber PG, Giardina PJV (1982) Linkage of betathalassaemia mutations and beta-globin gene polymorphisms with DNA polymorphismsin human beta-globin gene cluster. Nature (London) 296:627-631. Rodrigues NR, Rowan A, Smith MEF, Kerr IB, Palmer WF, Gannon JV, Lane DP (1990) p53 mutations in colorectal cancer. Proc Natl Acad Sci USA 87:7555-7559.

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Rossiter BJF, Caskey CT (1990) Molecular scanning methods of mutation detection. J Biol Chem 265:12753-12756. Saleeba JA, Cotton RGH (1991) 35S-labelledprobes improve detection of mismatched base pairs by chemical cleavage. Nucleic Acids Res 19:1712. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual. New York: Cold Spring Harbor Press. Spritz RA, Jagadeeswaran P, Choudary PV, Biro PA, Elder JT, deRiel JK, Manley JL, Gefter ML, Forget BG, Weissman SM (1981) Base substitution in an intervening sequence of a beta -thalassemic human globin gene. Proc Natl Acad Sci USA 78~2455-2459. Trecartin RF, Liebhaber SA, Chang JC, Lee KY, Kan YW, Furbetta M, Angius A, Cao A (1981) Beta zero thalassemia in Sardinia is caused by a nonsense mutation. J Zlin Invest 68: 1012-1017. Westaway D, Williamson R (1981) An intron nucleocide sequence variant in a cloned beta + -thalassemia globin gene. Nucleic Acids Res 9:1777-1788.

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Complete mutation detection using unlabeled chemical cleavage.

We have developed a strategy for the complete detection of point mutations, small insertions and deletions by chemical cleavage based on the methodolo...
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