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Automated fluorescent analysis procedure for enzymatic mutation detection.

The requirement for a highly sensitive, robust mutation detection assay has resulted from the rapid advances in the field of molecular genetics. In particular, the large amount of sequence data made available through the Human Genome Project and the expeditious discovery of new genes has resulted in a need for improved DNA-scanning methods (3, 4). High-throughput analysis of genetic variants requires a mutation detection method that uses conventional equipment and standard molecular techniques and is simple, fast, and robust. The method should be able to screen large stretches of DNA without reducing diagnostic sensitivity or specificity, at the same time providing information about the location and nature of the mutation. No commonly used methods possess all of these key attributes.

Of the numerous methods devised in the past 20 years for detecting mutations (5), the most widely used are single stranded conformation polymorphism (SSCP) (6, 7) and automated DNA sequencing (8). SSCP is favored because of its simplicity and its low cost, whereas automated DNA sequencing has the advantage of providing improved sensitivity and specificity. Unfortunately, the sensitivity of SSCP is highly variable; it detects only 60-80% of mutations and requires optimization of conditions for each amplicon tested (9). DNA sequencing, on the other hand, is highly sensitive (>95%) in the detection of homozygous mutations but suffers from high costs and tedious data analysis. Fluorescent sequencing can have variable sensitivity and specificity in detecting heterozygotes because of the inconsistency of base-calling at these sites (10).

Other methods, including denaturing gradient gel electrophoresis (11) and chemical cleavage of mismatch (12) have been used with some success. However, despite their sensitivity improvements over SSCP, these methods are technically challenging and have not been widely adopted. Use of heteroduplex analysis, which relies on the heteroduplexes formed after the hybridization of mutant and wild-type DNA possessing differing mobilities in nondenaturing gels, has been limited to detection of insertions/ deletions (13). Detection of single base pair mismatches is problematic with this method, as is detection of unknown mutations. Additionally, heteroduplex analysis does not provide the location of the mutation.

We developed Enzymatic Mutation Detection (EMD) to solve the problems of existing methods for detecting mismatch structures in duplex DNA. EMD uses a resolvase enzyme cloned from bacteriophage [T.sub.4], known as endonuclease VII ([T.sub.4] endo VII) (14). In vivo, [T.sub.4] endo VII cleaves branched DNA intermediates that form during phage DNA replication and packaging (15). An additional feature of the resolvase class of enzymes is the ability to cleave DNA duplexes at sites containing mispaired DNA (16). For the purposes of the EMD assay, the key structures recognized by the resolvase are "bubbles" formed by pairing wild-type DNA with a sample containing a point mutation and "heteroduplex loops" formed by hybridizing wild-type DNA with a sample containing an insertion or deletion.

In the EMD assay, the [T.sub.4] endo VII enzyme scans along double-stranded DNA until it detects a structural distortion caused by single base pair mismatches and cleaves within 6 by on the 3' side of the mutation (16). The double-stranded DNA fragments resulting from resolvase cleavage can be detected by conventional analytical methods such as gel electrophoresis. EMD is a single-tube assay that requires PCR amplification of the DNA of interest, formation of heteroduplex DNA, enzymatic mismatch cleavage, and analysis by gel electrophoresis (Fig. 1).

[FIGURE 1 OMITTED]

Materials and Methods

DNA ISOLATION

Isolation of plasmid and genomic DNA was performed according to established methods (17,18). Amplified PCR products were provided by collaborators for use in additional PCR amplifications. DNA was diluted in 10 mM Tris (pH 8)-1 mM EDTA buffer (TE) to 100 ng/[micro]L in preparation for DNA amplification.

EMD ASSAY FOR HOMOZYGOUS MUTATIONS

PCR amplification of reference and test DNA. Various templates (plasmid, genomic, or amplified PCR products) were amplified with 6-carboxyfluorescein-labeled primers (Perkin-Elmer-Applied Biosystems) under standard PCR conditions [e.g., 100 ng of template; 1X PCR buffer consisting of 50 mmol/L KCI, 10 mmol/L Tris-HCI, pH 8.3,1.5 mmol/L Mg[Cl.sub.2], 800 [micro]mol/L of dNTPs (2 mmol/L each), 150 nmol/L of primers, 1.5 U AmpliTaq Gold[R], and sterile water to 50 [micro]L; amplifications at 95 [degrees]C for 12 min, 94 [degrees]C for 30 s, 55-65 [degrees]C for 1 min (annealing temperature is template-dependent), and 72 [degrees]C for 30 s for 30 cycles, and at 72 [degrees]C for 12 min on a PE 9600 Thermocycler (Perkin-Elmer)]. A portion of the reference, or wild-type, DNAs were purified with Centricon[R] 50 (Amicon) concentrators and used as probes in the assay for heteroduplex formation. The wild-type, or reference, DNA (unpurified) was also used as a homoduplex control. All amplified test DNAs were used directly in the assay without any purification.

Hybridization. The hybridization reaction consisted of mixing 10 [micro]L of amplified test DNA with 2 [micro]L of hybridization buffer (EMD Fluorescent Kit, Avitech) and 3 [micro]L of probe (consisting of 100 fmol of purified wild-type DNA diluted in TE). The reactions were incubated in a heat block at 95 [degrees]C for 5 min, then at room temperature for 5 min.

Enzyme detection. Five microliters of diluted [T.sub.4] endo VII [1000 U per reaction; 2 [micro]L of enzyme and 3 [micro]L of enzyme dilution buffer (EMD Fluorescent Kit, Avitech)] was added to each 15-[micro]L hybridization reaction. The reactions were incubated in a heat block at 37 [degrees]C for 30 min. Each reaction was stopped by adding 30 [micro]L of Stop Mix, which consisted of 5 [micro]L of Perkin-Elmer-Applied Biosystems GeneScan-500[TM] TAMRA, 5 [micro]L of Perkin-Elmer-Applied Biosystems loading buffer, and 20 [micro]L of formamide.

Electrophoretic analysis. Twelve-centimeter 6% Long Ranger[TM] (FMC BioProducts) sequencing gels with well-forming combs and containing 6 mol/L urea and 0.5X Trisborate-EDTA were used. Gels were prerun at 750 V, 35 mA, 50 W, and 51 [degrees]C for 15 min on the ABI Prism[TM] 377 Sequencer equipped with GeneScan[TM] software (Perkin-Elmer-Applied Biosystems). Before loading, samples were denatured in a heat block at 95 [degrees]C for 3 min and placed on ice. Each sample (2.5 [micro]L) was loaded onto the gel, which was run at 750 V, 60 mA, 200 W, and 51 [degrees]C for ~2 h. Data analysis was performed on the ABI with GeneScan software.

EMD ASSAY FOR HETEROZYGOUS MUTATIONS

A gene that contains one normal allele and one potentially mutant allele does not require the addition of reference (wild-type) DNA. The test DNA is amplified with fluorescently labeled forward and reverse primers. During the hybridization step, DNA from the normal allele is denatured and annealed to the potentially mutant allele. If a mutation is present, heteroduplex structures will be formed during hybridization and cleaved by [T.sub.4] endo VII.

PCR amplification of reference and test DNA. BRCA1 genomic templates were amplified with FAM-labeled primers under standard PCR conditions such as those noted above. Because one normal allele and one potentially mutant allele was present in these samples, the preparation of a reference probe for heteroduplex formation was unnecessary. The wild-type, or reference, DNA was used as a homoduplex control. The amplified reference and test DNA were used as unpurified PCR products in the assay.

Hybridization. The hybridization reaction consisted of mixing 10 [micro]L of amplified test DNA with 2 [micro]L of hybridization buffer and 3 [micro]L of TE. The reactions were incubated in a heat block at 95 [degrees]C for 5 min, then at room temperature for 5 min. The remainder of the procedure is identical to the steps in the homozygous mutation assay.

Results

Representative electropherograms from the mouse [beta]-globin promoter gene and an amplified portion of the human p53 gene that have undergone EMD analysis are shown in Fig. 2. The wild-type sample in each electropherogram is shown in black and the test samples are in red. In each case, the EMD method identifies the site of mutation by sizing the two fragments on the GeneScan analysis software. The two samples from the mouse [beta]-globin gene showed cleavage products at 101 and 459 by (first electropherogram), and 60 and 500 by (second electropherogram). The two samples from the human p53 gene showed cleavage products at 236 and 244 by (third electropherogram) and at 200 and 280 by (fourth electropherogram). The fluorescent signal shown at -40 by and below is the result of labeled PCR primers. Fig. 2 represents typical results from the EMD assay system.

ASSAY SENSITIVITY STUDY

To determine the clinical importance of an assay procedure, it is critical to test the method with blinded samples that include both positive and negative samples. For this study, a collaborator supplied 92 unknown p53 samples obtained from breast cancer patients. Automated cDNA sequencing by our collaborator identified mutations in 22 of the samples, and scored 70 samples as being wild-type. Using the fluorescent EMD method, we identified 26 mutant samples correctly and scored 66 samples as wild-type, giving 100% sensitivity and 94% specificity compared with sequencing results (Table 1). The predicted value of a positive test is 84.6%, and the value of a negative test is 100% when compared with sequencing.

ASSAY RANGE OF DETECTION

A robust mutation detection method requires that an assay be capable of accepting a broad range of test DNA concentrations, which is typical for most laboratory PCR products. To identify an operating range for the EMD assay, we varied the concentrations of both amplified wild-type and mutant DNA. Thus, equal concentrations of purified mutant and wild-type DNA were mixed in the hybridization reaction of each assay. Fig. 3A shows a plot of the DNA concentration vs mutant cleavage signal (represented by peak areas) derived from an ABI 377 electropherogram where 2 pmol of wild-type DNA was mixed with 2 pmol of mutant DNA in a 10-[micro]L reaction volume, followed by 1.5 pmol of wild-type DNA mixed with 1.5 pmol of mutant DNA, 1 pmol of wild-type DNA mixed with 1 pmol of mutant DNA, 0.5 pmol of wild-type DNA mixed with 0.5 pmol of mutant DNA, and 0.1 pmol of wild-type DNA mixed with 0.1 pmol of mutant DNA. The linear response curve shown here allowed the detection of a 20-fold dynamic range of PCR concentrations.

[FIGURE 2 OMITTED]

ASSAY LIMIT OF DETECTION

To determine the limit of detection for the EMD assay, we altered the volume of amplified mutant (test) DNA in the reaction mixture. For this purpose, a constant amount of wild-type DNA and varying amounts of mutant DNA (diluted with increasing volumes of PCR buffer, 0-9 [micro]L) were used. Fig. 3B shows a plot of the amount of mutant DNA vs peak area, with the highest volume of mutant DNA being 10 [micro]L, followed by 5 [micro]L of mutant DNA diluted with 5 [micro]L of PCR buffer, 2.5 [micro]L of mutant DNA diluted with 7.5 [micro]L of PCR buffer, and 1 [micro]L of mutant DNA diluted with 9 [micro]L of PCR buffer. Even when the mutant sample was present at one-tenth of its initial volume (lowest point on the curve), the cleavage signal was readily detected (i.e., peak area >1000 fluorescent units). Because the assay can easily detect a broad range of mutant DNA concentrations, quantitating the PCR products is not necessary.

[FIGURE 3 OMITTED]

MIXED SAMPLE AND HETEROZYGOTE SENSITIVITY

For a mutation detection method to be a useful analytical tool, it must be capable of detecting mismatches down to 5-10%, because the method may be required to detect low concentrations in mixed samples. In both clinical and research settings, samples frequently contain mixtures of normal and mutant DNA. The most common example is a heterozygote, where the individual contains both a normal and mutant allele. Another example is DNA extracted from tumor samples. Thus, sufficient sensitivity to analyze mixed samples is critically important in a test method.

This experiment was performed to show the power of EMD in detecting mutations even when they are a small fraction of the total sample. A BRCA1 exon 11 (480 bp) genomic DNA sample was used as the starting material for this study.

In Fig. 4, varying amounts of both amplified mutant and wild-type DNA were mixed before the hybridization reaction. Because the mutant DNA is from a heterozygous gene, heteroduplex formation occurs and cleavage is detected without the addition of wild-type DNA (the 10-[micro]L mutant volume shown in Fig. 4). Increasing the amount of wild-type DNA (i.e., 7.5 [micro]L of mutant:2.5 [micro]L of wild-type, 5 [micro]L of mutant:5 [micro]L of wild-type, 2.5 [micro]L of mutant:7.5 [micro]L of wild-type, and 1 [micro]L of mutant:9 [micro]L of wild-type) shows that mutation detection in a mixture containing as little as 5% of the test sample is possible (i.e., 5% because of the heterozygous nature of the sample).

[FIGURE 4 OMITTED]

ASSAY REPRODUCIBILITY

For an assay technique to be useful as a precise and accurate clinical analysis tool, the procedure must be capable of providing reproducible results. Three p53 PCR samples were amplified in five independent PCR reactions, and the EMD assays were carried out on each sample over three consecutive days by three separate investigators (Table 2). The mutation was consistently identified each time, and the average percentage of the CV associated with the mean peak area is 9.9%, which is equivalent to the instrument precision (data not shown). This shows excellent reproducibility for both research and clinical assays.

RANGE OF FRAGMENT SIZES

To show that the EMD assay can efficiently detect mutations in fragments larger than 1 kb, we obtained four differently sized amplicons from exon 11 of the BRCA1 gene. A set of four fragments covering the same mutation and varying in size from 267 to 1070 by was generated for this gene (Fig. 5). Because only one primer was labeled in this sample set, only one fragment was detected in each test sample. The same forward primer was used in each experiment. This forward primer was paired with four different reverse primers, starting at the position closest to the forward primer and moving to the next downstream reverse primer for each successive PCR amplification. The EMD assay was then carried out on each fragment.

The PCR reactions for this study were not optimized; multiple PCR products were observed for each amplicon in an acrylamide check gel (data not shown). Hence, an increased background signal can be seen, particularly in the larger fragment sizes (763 and 1070 bp). The 200-bp band corresponding to the mutation is clearly detected even with substantial background noise.

[FIGURE 5 OMITTED]

Discussion

Applications of mutation detection are expanding rapidly in areas such as functional genomics, clinical diagnostics, and molecular profiling of participants in clinical trials. Current methods require users to accept suboptimal performance in one or more key areas, including sensitivity, specificity, reliability, or ease of use. We have shown that the EMD assay based on [T.sub.4] endo VII optimizes each of these factors.

Relative to SSCP the greatest advantage of EMD is a dramatic improvement in sensitivity, from 60-80% to near 100%. Unlike SSCP, which must be optimized for each amplicon tested, a single EMD protocol may be used for all amplicons. EMD analyzes larger fragments than SSCP (sim]1200 vs ~250 bp), and reactions can be performed easily on a robotic pipetting station.

Relative to DNA sequencing, EMD offers substantial improvements in simplicity, throughput, and cost. Typically, amplicons from 200-1200 by can be analyzed with the EMD fluorescent assay on the ABI 377, which reduces the total number of amplicons necessary to analyze a given exon. Fluorescent DNA sequencing typically requires amplicons no larger than 400 by for accurate reads. Moreover, EMD is considerably more sensitive than DNA sequencing in the detection of heterozygous samples (Fig. 4). As a result, detection of mutations in the mixed samples typical of tumor biopsies is possible with EMD, whereas it is often impossible with DNA sequencing (19), as was evident from the results of the unknown p53 sample analysis (Table 1). It should be noted that the lower specificity compared with sequencing seen in Table 1 is believed

to be the result of the superior sensitivity of EMD in detecting mutations in heterogeneous DNA, which is characteristic of tumor samples.

Because EMD scanning does not identify the specific base changes that occur in a mutation or polymorphism, positive samples may require confirmation by direct DNA sequencing. A major advantage of EMD is that the equipment, biochemistry, and work flow for EMD analysis is entirely compatible with the Sanger method of sequencing (20). In a completely manual assay mode, a single technician can process 50 EMD reactions per day (including PCR amplification and gel electrophoresis). This number increases to 120 per day when a single ABI sequencer with multipipettes and 96-well microplates is used. Adding a pipetting station to this workstation improves the throughput to 400 reactions per day. The limit then becomes the number of gels that can be run (per ABI) in a single 8-h shift (we are currently running 3 gels per 8-h day).

Although [T.sub.4] endo VII can detect all possible mismatches, it does so with differing affinities (21). Specifically, G-containing mismatches are less preferred than C-containing mismatches. The EMD assay has been designed to take advantage of the two distinct heteroduplex structures that are created when a reference DNA is hybridized to a mutant sample (Fig. 1). For example, a fragment containing an AT pair at a particular site, when hybridized with a CG pair in a mutant fragment, yields an AG-containing heteroduplex as well as a TC-containing heteroduplex. The latter is more strongly detected (manuscript in preparation). The typical cleavage pattern is one strong band (TC) and one weaker band (AG) (1, 2, 22). In the optimized EMD fluorescent assay, both cleavage peaks have been detected in 95% of the samples tested to date, whereas a single peak has been detected in only 5% of the samples (data not shown).

A key criterion for assay success is the quality of the PCR reactions. In particular, mispriming that reduces PCR fidelity creates fragments of variable lengths that could become substrates for nonspecific enzyme cleavage (Fig. 5). Background problems can be avoided by optimizing PCR conditions before proceeding with EMD (23). Although EMD works well with standard Taq polymerase, we have achieved the best results with plasmid and genomic templates by using AmpliTaq Gold, a mutant Taq polymerase that is active only at 95 [degrees]C, thus providing the improved fidelity of "hot start" PCR (24, 25).

In summary, the EMD assay is a simple, four-step, single-tube procedure that does not require optimization for different substrates; therefore, a single protocol may be used. The procedure is automated, detects mutations in long fragments up to 4 kb, and is performed in <1 h (excluding gel electrophoresis). All mismatch types are detected, including insertions and deletions. The assay offers particularly high sensitivity for heterozygotes. The assay is robust, and the data are highly reproducible. The assay method uses the conventional equipment and procedures available in most molecular biology and diagnostics laboratories, but it is adaptable to new analytical methods. Lastly, EMD reactions do not require purification of test samples.

We thank Pharmacia Biotech AB for providing clinical p53 PCR products and the cDNA sequencing results reported for the independent evaluation. We also thank Mary K. Luke for her assistance in the preparation of this manuscript and its illustrations.

References

(1.) Youil R, Kemper B, Cotton RGH. Detection of 81 of 81 known mouse R-globin promoter mutations with [T.sub.4] endonuclease VII-the EMC method. Genomics 1996;32:431-5.

(2.) Youil R, Kemper B, Cotton RGH. Screening for mutations by enzyme mismatch cleavage with [T.sub.4] endonuclease VII. Proc Natl Acad Sci U S A 1995;92:87-91.

(3.) Cotton RGH. Current methods of mutation detection. Mutat Res 1993;285:125-44.

(4.) Kwok P-Y, Carlson C, Yager TD, Ankener W, Nickerson DA. Comparative analysis of human DNA variations by fluorescence-based sequencing of PCR products. Genomics 1994;23:138-44.

(5.) Cotton RGH. In: Cotton RGH, ed. Mutation detection. Oxford: Oxford University Press, 1997:177-93.

(6.) Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as singles-trand conformation polymorphisms. Proc Natl Acad Sci U S A 1989;86:2766-70.

(7.) Selvakumar N, Ding B-C, Wilson S. Separation of DNA strands facilitates detection of point mutations by PCR-SSCP. Biotechniques 1997;22:604-6.

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(9.) Glavac D, Dean M. Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations. Hum Mutat 1993;2:404-14.

(10.) Nickerson D, Tobe V, Taylor S. PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res 1997;25: 2745-51.

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(12.) Smooker PM, Cotton RGH. The use of chemical reagents in the detection of DNA mutations. Mutat Res 1993;288:65-77.

(13.) Cotton RGH. In: Cotton RGH, ed. Mutation detection. Oxford: Oxford University Press, 1997:58-67.

(14.) Kosak HG, Kemper BW. Large-scale preparation of [T.sub.4] endonuclease VII from over-expressing bacteria. Eur J Biochem 1990;194: 779-84.

(15.) Kemper B, Garabett M. Studies on [T.sub.4] -head maturation. 1. Purification and characterization of gene-49-controlled endonuclease. Eur J Biochem 1981;115:123-31.

(16.) Solaro P, Birkenkamp K, Pfeiffer P, Kemper B. Endonuclease VII of phage [T.sub.4] triggers mismatch correction in vitro. J Mol Biol 1993; 230:868-77.

(17.) Bimboin GC, Daly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 1979;7: 1513-22.

(18.) Sambrook J, Fritsch EF, Maniatis T. Purification of nucleic acids. In: Ford N, Nolan C, Ferguson M, Ockler M, eds. Molecular cloning: a laboratory manual, 2nd ed. Plainview: Cold Spring Harbor Laboratory Press, 1989:E3-E7.

(19.) Cheng J, Haas M. Sensitivity of detection of heterozygous point mutations in p53 cDNAs by direct PCR sequencing. PCR Meth and Appl 1992;1:199-201.

(20.) Sanger F, Nicklen S, Couson AR. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463-7.

(21.) Pottmeyer S, Kemper B. [T.sub.4] Endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence. J Mol Biol 1992;223:607-15.

(22.) Mashal RD, Koontz J, Sklar J. Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 1995;9:177-83.

(23.) Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res 1997;25:3235-41.

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(25.) Chou Q, Russell M, Birch DE, Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res 1992;20:1717-23.

BENJAMIN J. DEL TITO, JR., [1], [2] * HERBERT E. POFF III, [1] [2] MARK A. NOVOTNY, [1] [2] DONNA M. CARTLEDGE, [1] [2] RALPH I. WALKER II, [1] [2] CHRISTOPHER D. EARL, [1] and ANNE L. BAILEY [1], [2] [dagger]

[1] Avitech Diagnostics, Inc., 30 Spring Mill Drive, Malvern, PA 19355. [2] Variagenics, Inc., One Kendall Square, Cambridge, MA 02139.

* Present address: North American Vaccine, Inc., 12103 Indian Creek Court, Beltsville, MD 20755.

[dagger] Address correspondence to this author at: North American Vaccine, Inc., 12103 Indian Creek Court, Beltsville, MD 20705.

Received November 5, 1997; revision accepted December 23, 1997.
Table 1. Blinded clinical study of the p53 tumor suppressor
gene comparing EMD with DNA sequencing analysis.

 cDNA sequencing EMD (a)

Mutant 22 26
Wild-type 70 66

Total 92 92

(a) Percent sensitivity and specificity for the EMD
method equivalent to 100% and 94%, respectively.

Table 2. EMD assay reproducibility using three separate
p53 samples analyzed by three investigators on separate
days.

 Sample
 1

Investigator Cleavage 1 (a) Cleavage 2

1 6659 [+ or -] 534 21 829 [+ or -] 1211
2 5887 [+ or -] 529 18 773 [+ or -] 1472
3 7514 [+ or -] 541 19 883 [+ or -] 1575
CV (b) 8.0 7.0

 Sample
 2

Investigator Cleavage 1 Cleavage 2

1 8050 [+ or -] 978 1960 [+ or -] 434
2 7675 [+ or -] 573 2800 [+ or -] 230
3 6844 [+ or -] 859 1219 [+ or -] 458
CV (b) 10.7 18.8

 Sample
 3

Investigator Cleavage 1 Cleavage 2

1 4511 [+ or -] 211 4421 [+ or -] 755
2 5345 [+ or -] 211 5351 [+ or -] 426
3 2406 [+ or -] 102 3049 [+ or -] 203
CV (b) 4.3 10.8

(a) Mean peak area [+ or-] SD associated with each
cleavage peak (n = 3).

(b) Percentage for each cleavage peak (n = 9).
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Title Annotation:Molecular Pathology and Genetics
Author:Del Tito, Benjamin J., Jr.; Poff, Herbert E., III; Novotny, Mark A.; Cartledge, Donna M.; Walker, Ra
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Apr 1, 1998
Words:4196
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