Microsatellite Acrylamide

Fig. 10. Detection of mutations and sequence variants by heteroduplex analysis of the CFTR gene. Exon 10 of the CFTR gene was amplified by PCR for heteroduplex analysis. The gel is 1.5 mm thick and 40 cm long 1X MDE (Cambrex Bio Science Walkersville, Inc.) containing 15% urea. The gel was run for 21,000 V-h in 0.6X Tris-borate-EDTA buffer and stained with ethidium bromide. Lanes 1 and 13 are size markers (100 bp ladder), lane 2 is a MM homozygote at postion 470, lane 3 is a VV homozygote at position 470, lane 4 is a heterozygote for the M470V polymorphism, lane 5 is a homozygote for the AF508 mutation, lane 6 is a AI507/wild-type heterozygote, lane 7 is a compound heterozygote for AF508 and I506V, lane 8 is compound heterozygote for AF508 and F508C, lane 9 is a compound heterozygote for AF508 and Q493X, lane 10 is an I506V/wild-type heterozygote, lane 11 is a F508C/wild-type heterozygote, and lane 12 is a AF508/ wild-type heterozygote.

of heteroduplexes vs homoduplexes in a set of constructs similar to the DNA Toolbox described earlier (35). The PCR products ranged in size from 153 to 938 bp. A spike in resolution between heteroduplexes and homoduplexes was observed between 150 and 200 bp, with the resolution decreasing with increasing fragment size. However, sufficient resolution remained at 500 bp to be easily detectable. The greatly enhanced separations seen near 200 bp of fragment length were explained in terms of the model of DNA electrophoresis of Calladine et al. (36), in which DNA is seen as a superhelix with inherent flexibility and a pitch of approx 200 bp. According to this interpretation, in longer DNA fragments, electrophoretic mobility is more influenced by bending than it is by local structural changes induced by mismatches.

One advantage of CSGE is the reproducibility of the patterns obtained with specific mutations. This can be of great help in the repetitive analyses of genes that have multiple benign polymorphisms as well as disease-causing mutations. Once the patterns of common polymorphisms are cataloged, there is no need to reflex samples exhibiting these patterns to sequencing. Figure 11 shows a typical CSGE gel interrogating three exons of the RET proto-oncogene in a screen for multiple endocrine neoplasia. A second advantage of CSGE over heteroduplex analysis on MDE gels is that the reagents are not proprietary. It would be of substantial interest to see a study comparing the two techniques for sensitivity and specificity of mutation detection. An advantage that CSGE and HA share over SSCP is that only one condition is needed and gels do not have to be run at subambient temperatures (i.e., in a cold room).

5.4. MUTATION DETECTION USING DENATURING GRADIENT GEL ELECTROPHORESIS Denaturing gradient gel electrophoresis (DGGE) was one of the first scanning methods used for the identification of mutations in DNA (37). The method is based on the principle that the denaturation, or melting, of double-stranded DNA—by heat or denaturants such as hydroxide ion, urea, or formamide—does not occur in a single step; rather, DNA melts in domains. As the temperature rises or the denaturant concentration rises, the region or domain with the highest A + T content will melt first. If the temperature or the denaturant concentration is kept constant, the DNA structure composed of double-stranded DNA and a single region of single-stranded character will be stable. If the temperature is increased again, the region with the next highest A + T content will melt next. This melting by domain continues until the region with the highest G + C content is melted and the character of the DNA is completely single-stranded. As the identity of the melting domains is a function of the base sequence, a change in the base sequence (i.e., a mutation) will likely change the melting profile (Fig. 12). If the mutation does not alter the melting profile, it will not be detected. However, if a heterodu-plex is formed and then subjected to melting analysis, a change in melting profile will almost certainly be seen. Thus, for maximum sensitivity, DNA heteroduplexes are formed between a control DNA fragment of known sequence and the test DNA.

The melting profile of heteroduplex DNA is observed by electrophoresis on a transverse denaturing gradient gel. In this system, a polyacrylamide gel is poured containing a gradient of denaturant, typically urea and formamide. After polymerization, the gel is rotated 90°, the DNA heteroduplex sample is applied to a single troughlike well, and the electrophoresis is performed. The DNA migrating through the region of the gel with the lowest denaturant concentration migrates as typical DNA; the DNA migrating through the denaturant concentration corresponding to the first melting domain will migrate with significantly lower mobility; the DNA migrating through the region corresponding to the next melting domain will migrate slower still. This stepfunction of decreasing mobility continues until the highest melting-point domain denatures, yielding rapidly moving single-stranded DNA. After visualization of the DNA with ethidium bromide or silver, a stair-step pattern is seen. In the case of a heteroduplex sample, the domain containing

Mirosatelites Scoring Gel

Fig. 11. Detection of mutations and sequence variants by CSGE analysis of the RET oncogene. Exons 13, 14, and 16 of the RET oncogene were amplified in a multiplex reaction containing 33P-dATP. CSGE was performed as described by Ganguly (35); bands were detected by autoradiography. Lanes 9-14 are controls for common polymorphisms. Lane 9 is a heterozygote for the E768D polymorphism. Lane 10 shows both L769L in exon 13 and V804L in exon 14. Lane 11 shows a sample bearing two polymorphisms in exon 13: E768D and L769L. Lane 12 shows M918T in exon 16. Lane 13 shows V804M in exon 14. Lane 14 shows L769L in exon 13 and S836S in exon 14. Lanes 1-8 are patient samples. Lanes 1, 2, 5, 6, and 8 are positive for the common L769L polymorphism. There are no pathogenic mutations identified on this gel.

Fig. 11. Detection of mutations and sequence variants by CSGE analysis of the RET oncogene. Exons 13, 14, and 16 of the RET oncogene were amplified in a multiplex reaction containing 33P-dATP. CSGE was performed as described by Ganguly (35); bands were detected by autoradiography. Lanes 9-14 are controls for common polymorphisms. Lane 9 is a heterozygote for the E768D polymorphism. Lane 10 shows both L769L in exon 13 and V804L in exon 14. Lane 11 shows a sample bearing two polymorphisms in exon 13: E768D and L769L. Lane 12 shows M918T in exon 16. Lane 13 shows V804M in exon 14. Lane 14 shows L769L in exon 13 and S836S in exon 14. Lanes 1-8 are patient samples. Lanes 1, 2, 5, 6, and 8 are positive for the common L769L polymorphism. There are no pathogenic mutations identified on this gel.

Fig. 12. DNA melts in domains. When treated with heat or chemical denaturants (urea or formamide), double-stranded DNA does not melt all at once, rather it melts in domains.

the mismatch will melt early, yielding a characteristic doublet pattern. (Fig. 13).

Clearly, pouring gradient gels and analyzing samples one at a time is labor-intensive and time-consuming. Fortunately, the melting profile of any DNA fragment can be modeled mathematically. Computer programs are commercially available that calculate the melting profile for PCR-amplified DNA (38). Using this tool, gradients can be optimized for each piece of DNA. Using optimized gradients, gels can be run in a more conventional manner (i.e., with the electrophoresis driving the

Microsatellite Instability

Fig. 13. A transverse DGGE gel of exon 10 of the CFTR gene from an individual heterozygous for the AF508 mutation. The direction of electrophoresis is top to bottom and the denaturant concentration increases from left to right. The heteroduplex product melts at a lower temperature than the first domain of the homoduplexes. Thus, the mobility transition occurs further to the left (lower denaturant concentration) than for the homoduplexes, giving the characteristic "cats-eye" pattern, indicative of the presence of a mixture of sequences.

Fig. 13. A transverse DGGE gel of exon 10 of the CFTR gene from an individual heterozygous for the AF508 mutation. The direction of electrophoresis is top to bottom and the denaturant concentration increases from left to right. The heteroduplex product melts at a lower temperature than the first domain of the homoduplexes. Thus, the mobility transition occurs further to the left (lower denaturant concentration) than for the homoduplexes, giving the characteristic "cats-eye" pattern, indicative of the presence of a mixture of sequences.

DNA into ever higher denaturant concentrations). In this format, a sample bearing a low melting-point domain, such as a heteroduplex, will exhibit a band of slower mobility.

In theory, mutations in the highest melting-point domain will not be detected by DGGE because it is difficult to elec-trophoretically determine the exact point at which the transition from the slow migrating species (with the last melting-point domain intact) to the rapidly migrating single-stranded species occurs. In order to eliminate this caveat to the technique, a "GC clamp" is typically added to the amplified product by preparing one of the PCR primers with a 5' tail of 30-50 nucleotides of 100% G+C content. Thus, the artificial "GC clamp" becomes the highest melting-point domain, and mutations in all of the original domains can be detected (39). Although the sensitivity of DGGE for the detection of unknown mutations approaches 100%; the popularity of the method seems to be decreasing because of the expense of PCR primers that are 70-80 nucleotides in length, the difficulty in amplification with primers of such high-melting temperatures, and the difficulties associated with the need to freshly prepare reproducible gradient gels. However, an experimentally more facile system, temperature sweep or temporal thermal gradient gel electrophoresis (TTGE), was introduced by Yoshino et al. in 1991 (48). This system, which relies on the same principle as DDGE, is far more straightforward to carry out, as it does not involve pouring gradient gels. In a TTGE experiment, the denaturing gradient is provided as a smooth increase in temperature during the elec-trophoretic run. Wong et al. recently reported the use of TTGE

in a comprehensive scan of the entire mitochondrial genome from 179 patients for deleterious mutations and polymorphisms. (49). A system for TTGE, Dcode™, is commercially available form Bio-Rad (Hercules, CA).

5.5. MUTATION DETECTION BY CHEMICAL CLEAVAGE OF MISMATCHED NUCLEOTIDES The chemical cleavage of mismatches technique, described by Cotton in 1988, takes advantage of the differential reactivity of perfectly paired and mismatched bases to chemical modifying reagents (50). In het-eroduplex species in which a thymine nucleotide is mismatched, the T residue is hypersusceptible to chemical modification by osmium tetroxide (OsO4), a commonly used shadowing reagent for electron microscopy. Similarly, mismatched cytosine nucleotides are hypersusceptible to attack by hydroxlamine (HONH2). DNA strands containing either a modified T or C nucleotide are then cleaved with piperidine (C5HnN). In practice, the DNA to be screened for mutations is amplified and then mixed with a 5- to 10-fold molar excess of wild-type amplicon. This control DNA, referred to as the probe, is typically labeled on one strand with 32P. After mixing, melting, and reannealing, the resultant heteroduplexes are divided into two aliquots. One aliquot is treated with OsO4 and the other with HONH4. After treatment with these reagents, the samples are treated with piperidine and separated by electrophoresis on a sequencing-type polyacrylamide gel. If a mutation is present, it will be detected as an extra band after autoradiography. If the sample is tested twice (once with each strand of the probe DNA labeled), virtually 100% of all mutations will be detected. Furthermore, the exact position of the mutation can be defined by sizing the cleavage product. Sensitivity of CCM for the detection of mutations is very high (95-100%), the toxicity of the reagents, the large number of steps and manipulations, and the high background seen with many templates has limited the number of laboratories that have used this technique (reviewed in refs. 40 and 41).

5.6. MUTATION DETECTION BY RIBONUCLEASE CLEAVAGE OF MISMATCHED RNA : DNA DUPLEXES Mutation detection based on ribonuclease cleavage was developed when it was recognized that ribonucleases could cleave single-stranded RNA and that it was possible to synthesize radioactive RNA probes (42). RNA probes, or riboprobes, are synthesized using wild-type genomic DNA as a template with 32P incorporated as a label. The probe is hybridized to denatured target DNA to produce RNA : DNA hybrids. When there is a mismatch between the wild-type RNA probe and the DNA because of a mutation, the base or bases that have not annealed to the DNA are cleaved by RNAase A. The products of the digestion are denatured and separated by gel electrophoresis. A mutation is indicated by the presence of cleavage fragments of lower molecular weight than the full-length probe. The size of the cleavage products are used to determine the location of the specific mutation. Although this technique has been used to detect mutations in the hypoxanthine phosphoribosyltrans-ferase, type I collagen, and K-ras genes, it has not been widely employed partly because of the inability of RNAase A to completely cleave all mismatches (43-45). Single-stranded RNA beause of mismatches involving the purines adenine and gua-nine are not efficiently recognized by RNAase A. However,

Mismatch Chemical Cleavage

Fig. 14. Schematic representation of a nonisotopic RNase cleavage assay. The DNA or cDNA is amplified using primers with 5' T7 or SP6 promoter sequence to generate a template for in vitro transcription. The template, or target RNA, is hybridized with wild-type RNA to produce RNA : RNA duplexes. When a mismatch is present because of a mutation in the target RNA, the duplexes are cleaved by RNAase. The digested products are separated by gel electrophoresis and visualized by staining with ethidium bromide. (Courtesy of Marianna M. Goldrick, Ph.D., Ambion, Inc., Austin, TX.)

Fig. 14. Schematic representation of a nonisotopic RNase cleavage assay. The DNA or cDNA is amplified using primers with 5' T7 or SP6 promoter sequence to generate a template for in vitro transcription. The template, or target RNA, is hybridized with wild-type RNA to produce RNA : RNA duplexes. When a mismatch is present because of a mutation in the target RNA, the duplexes are cleaved by RNAase. The digested products are separated by gel electrophoresis and visualized by staining with ethidium bromide. (Courtesy of Marianna M. Goldrick, Ph.D., Ambion, Inc., Austin, TX.)

mismatches involving the pyrimidines cytosine and uracil or larger areas of single-stranded RNA (because of two mismatched sites in close proximity, a deletion or an insertion) are effectively cleaved. Modification and improvement of this method was made by incorporating PCR amplification of the target sequence and use of RNAase I (46). The starting template is either mRNA or genomic DNA. It is advantageous to use mRNA as a template because there are no intron sequences present. RNAase I recognizes all 4 bases when they are present at the site of a mismatch. However, like the original RNAase cleavage mismatch protocols, incomplete digestion of the hybrid molecules makes it difficult to distinguish between homozygotes and heterozygotes.

A nonisotopic RNase cleavage assay for mutation detection has been developed by Ambion, Inc., Austin, TX (47). Beginning with DNA or cDNA, the sequence to be screened is amplified using a forward primer with a T7 bacterial promoter sequence and a reverse primer with a SP6 bacterial promoter sequence (Fig.14). Target segments up to 1.0 kb long can be amplified and screened in one reaction. The added bacterial promoter sequences allow the PCR products to be transcribed in an in vitro system to produce large quantities of target RNA. The target RNA is hybridized with wild-type RNA to form

RNA : RNA duplexes. Because both template strands are transcribed, reciprocal mismatches (i.e., A-C and G-U) are created when each strand hybridizes to the wild-type RNA. This increases the likelihood that a mismatch will be cleaved because all sites are not cleaved with equal efficiency.

The hybrids are treated with RNAase, and any unpaired mismatched residues accessible to the enzyme are cleaved. The cleavage products are stained with ethidium bromide, separated by gel electrophoresis, and compared to the wild-type homod-uplex, which was also treated with enzyme. The wild-type homoduplex should be a single band of the highest molecular weight because it is resistant to cleavage. Although it is not possible to determine if the cleavage is at the 5' or 3' end of the target segment without rescreening, the size of the cleavage product does give a good estimation of the position of the mutation. A significant advantage of this method is the visualization of the cleavage products without radioactive probes. As shown in Fig. 15, this method can be used to screen for germline mutations in the breast and ovarian cancer susceptibility gene BRCA1. In addition to the detection of mutations, a nonisotopic Rnase cleavage assay (NIRCA) can also identify the genotype of a sample by hybridization of the sample to its own RNA transcripts. If a sample is heterozygous for a mutation,

Micro Satellite Instability

Fig. 15. Detection of mutations in the BRCA1 gene using a noniso-topic RNase cleavage assay. Target regions of the BRCA1 gene were amplified by nested PCR from genomic DNA from at-risk relatives of a familial breast cancer patient and from a normal control. The primers had promoter (T7 and SP6) sequences on the 5' ends. Crude PCR products (2 ||L) were transcribed with T7 and SP6 RNA polymerase. Complementary normal and test transcripts were mixed, heated briefly and cooled to make double-stranded RNA targets. Targets were treated with RNase for 45 min at 37°C, separated by electrophoresis through a 2% agarose gel, and then stained with ethidum bromide. Samples in lanes marked © were scored as positive for a putative mutation. The WT lane shows wild-type control sample. DNA size markers are indicated at the left margin. (Courtesy of Marianna M. Goldrick, Ph.D., Ambion, Inc. Austin TX.)

Fig. 15. Detection of mutations in the BRCA1 gene using a noniso-topic RNase cleavage assay. Target regions of the BRCA1 gene were amplified by nested PCR from genomic DNA from at-risk relatives of a familial breast cancer patient and from a normal control. The primers had promoter (T7 and SP6) sequences on the 5' ends. Crude PCR products (2 ||L) were transcribed with T7 and SP6 RNA polymerase. Complementary normal and test transcripts were mixed, heated briefly and cooled to make double-stranded RNA targets. Targets were treated with RNase for 45 min at 37°C, separated by electrophoresis through a 2% agarose gel, and then stained with ethidum bromide. Samples in lanes marked © were scored as positive for a putative mutation. The WT lane shows wild-type control sample. DNA size markers are indicated at the left margin. (Courtesy of Marianna M. Goldrick, Ph.D., Ambion, Inc. Austin TX.)

self-hybridization results in mismatched hybrids, which are cleaved. However, if a sample is homozygous, self-hybridization results in completely matched duplexes that are resistant to cleavage. The use of RNase cleavage methods for the detection of mutations has recently been reviewed by Goldrick (51).

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