Hetero Duplex Analysis

Fig. 8. Detection of p53 sequence variants by sscp analysis. A 183-bp fragment of exon 8 of the p53 gene was amplified from DNA extracted from four different breast tumor cell lines and human placenta (wild-type control). 33P-labeled dATP was included in the PCR to label the products that were separated by electrophoresis through a 0.5X MDE gel at room temperature. Lane 1: Wild-type control; lane 2, MDA-MB-231 cells; lane 3, MDA-MB-468 cells; lane 4, Bt-474 cells; lane 5 SkBr3 cells. The cell lines in lanes 2-4 contain mutations in exon 8 resulting in aberrant migration of the PCR products. (Courtesy of Lori Terry and J. Carl Barrett, Ph.D., Laboratory of Molecular Carcinogenesis, NIEHS.)

Fig. 8. Detection of p53 sequence variants by sscp analysis. A 183-bp fragment of exon 8 of the p53 gene was amplified from DNA extracted from four different breast tumor cell lines and human placenta (wild-type control). 33P-labeled dATP was included in the PCR to label the products that were separated by electrophoresis through a 0.5X MDE gel at room temperature. Lane 1: Wild-type control; lane 2, MDA-MB-231 cells; lane 3, MDA-MB-468 cells; lane 4, Bt-474 cells; lane 5 SkBr3 cells. The cell lines in lanes 2-4 contain mutations in exon 8 resulting in aberrant migration of the PCR products. (Courtesy of Lori Terry and J. Carl Barrett, Ph.D., Laboratory of Molecular Carcinogenesis, NIEHS.)

specialty matrices, such as MDE (21). Currently, many large laboratories run SSCP gels under several electrophoretic conditions (e.g., at room temperature and at 4°C, and with gels containing 0% or 10% glycerol) The use of multiple-run conditions can raise the sensitivity of the technique to virtually 100% (22).

Single-stranded conformational electrophoresis can be performed using CE as well as in the slab gel format. Although, at this writing, CE-SSCP has not been as thoroughly examined as SSCP using slab gels, several conclusions are beginning to emerge. As in slab gels, it appears that the fragment length is important in determining sensitivity of mutation detection, with longer fragments yielding decreased sensitivity (23). The role of

G + C content has not yet been examined in CE-SSCP. A wide variety of water-soluble, linear polymers have been used for CE-SSCP; however, there is yet to emerge a consensus as to which one is optimal. Because single-stranded DNA has more hydrophobic character than the double-stranded form, polymer hydrophobicity might prove to have a role in CE-SSCP separations. To date, only a single study has appeared examining the size of the polymer as a factor influencing mutation-detection sensitivity; that study (24) varied the molecular weight of linear polyacrylamide as the sieving matrix using the Molecular Dynamics MegaBase capillary array instrument (Sunnyvale, CA). The investigators compared linear polyacrylamides of molecular weight betweem 200 and 2400 kDa. They found that the lowest-molecular-weight solutions did not offer sufficient resolving power, whereas solutions of a 1000-kDA polymer gave good results. Increasing the polymer size to 2.4 x 106 Da offered no advantage; rather, very large polymers gave long analysis times without improved sensitivity and were difficult to load and flush from the capillary because of their very high viscosity.

Specific advantages of CE as a platform for SSCP include the high throughput and reproducibility. Investigators at the National Institute of Standards and Technology (NIST) have reported standard deviations of retention times in the range 0.05-0.5%. (25,26) By utilizing multiple fluors, multi-capillary array instruments, and the multiwavelength fluorescence detectors, extremely high throughout can be achieved. Software for the interpretation (mutation calling) in an automated fashion will be key to implementing high-throughput mutation scans using SSCP. Such a piece of software has been recently introduced by Hayashi's group. The program QUISCA accomplishes tasks such as high-frequency noise filtering, baseline correction, peak calling, and peak quantification; it is freely available to academic users (27).

Although the sensitivity of CE-SSCP can approach 100%, the specificity of the technique (indeed, of virtually all mutation detection strategies) is much less studied. Investigators at NIST reported 3 false positives in an analysis of 15 samples, a disturbingly high percentage (28). Others have reported false positives as a result of reannealing of PCR primers to the single-stranded DNA and the appearance of artifactual peaks (29). Clearly, the rate of false positives in genetic screening is an issue that needs to be addressed.

5.3. HETERODUPLEX ANALYSIS IN MUTATION DETECTION Heteroduplex formation is required for many mutation scanning methods. Heteroduplex analysis (HA) refers to the technique based on the electrophoretic resolution of wild-type double-stranded DNA fragments from fragments of identical length and sequence, but having one basepair mismatch. The mismatch is formed when a wild-type DNA fragment is mixed with a mutant DNA fragment. When the mixture is heated to denature the double-stranded material and allowed to cool to reanneal the single-stranded molecules, four types of molecule result: (1) wild-type DNA, formed when the Watson strand of the wild-type reanneals with the Crick strand of the wild-type; (2) mutant DNA, formed when the Watson strand of the mutant reanneals with the Crick strand of the mutant; and (3) two heteroduplex species, formed when the Watson strand of the wild-type reanneals with the Crick strand of the mutant and when the Watson strand of the mutant reanneals with the Crick strand of the wildtype (Fig. 9).

Two types of heteroduplex exist, the structures of which have been predicted by Bhattacharyya and Lilley (30). The first type is formed when the sequence difference between the two DNA fragments is one or more point mutations; the resulting heteroduplex is termed a "bubble"-type heteroduplex. The second type is formed when the sequence difference between the two fragments is a small insertion or deletion; the resulting heteroduplex is termed a "bulge"-type heteroduplex. Although "bulges" result in a large structural perturbation from the double-stranded homoduplex and are readily resolvable on poly-acrylamide gels, the change in overall structure as a result of a "bubble" is much more subtle, and these heteroduplexes typically are not resolvable from the homoduplexes on agarose or polyacrylamide gels run under standard conditions (see Subheading 5.3.2).

5.3.1. Heteroduplex Analysis on MDE™ Gels In 1991, a communication from Bhattacharayya's laboratory reported the superior separation of heteroduplex species on Hydrolink D-5000 (AT Biochem) (31). (Note: This gel matrix is no longer commercially available). As the proprietary formulation of D-5000 had not been developed for increased resolution of heteroduplexes, the results were a surprise to the scientists at AT Biochem who developed it. However, they quickly responded to the challenge of reformulating the material for optimum heteroduplex resolution. The resulting product, MDE™ (Mutation Detection Enhancement), has made HA analysis a viable technique for mutation detection. The development and properties of the Hydrolink series of gel matrixes have been described (32). Figure 10 shows heteroduplex analysis of exon 10 of the CFTR gene. The samples with mutations and sequence variants are characterized by the presence of extra bands with aberrant migration.

As with SSCP, there is no theory that can be derived to predict which mutations will be detected by HA and which ones

Heteroduplex Analysis

Fig. 9. Schematic representation of heteroduplex analysis. In HA mutations are detected by structural perterbations in the double-stranded DNA duplex because of the presence of one or more mismatches. Heteroduplexes as a result of single-base mismatches are not detected with high sensitivity on agarose or polyacrylamide, but they are typically resolved on specialty matricies such as MDE (Cambrex Bio Science Walkersville, Inc.).

Fig. 9. Schematic representation of heteroduplex analysis. In HA mutations are detected by structural perterbations in the double-stranded DNA duplex because of the presence of one or more mismatches. Heteroduplexes as a result of single-base mismatches are not detected with high sensitivity on agarose or polyacrylamide, but they are typically resolved on specialty matricies such as MDE (Cambrex Bio Science Walkersville, Inc.).

will not. In order to investigate the parameters affecting detection sensitivity of HA on MDE gels, Highsmith et al. conducted a study using the previously described DNA Toolbox. In contrast to SSCP, the G+C content of the DNA was not a significant parameter; rather, the detection sensitivity, over a range of PCR sizes from 100 to 600 bp, was most influenced by the specific mismatch. The detection rates and separation distances between heteroduplexes and homoduplexes generally followed the known thermodynamic instability of the mismatches, with G : G, C : C > A : G, T : C = T : G, A : G > A : A, T : T (33).

5.3.2. Conformation-Sensitive Gel Electrophoresis

Conformation-sensitive gel electrophoreis (CSGE) is a het-eroduplex analysis technique described by Ganguly and colleagues in 1993 (34). CSGE utilizes a polyacrylamide matrix with a novel crosslinker, bis-acrolylpiperazine (BAP). In addition, the CSGE matrix contains low concentrations of denaturing reagents. The concentration of these reagents is insufficient to cause complete separation of the DNA strands, but it is thought to further destabilize the conformational perturbations in the double-stranded DNA structure introduced by the presence of mismatched bases.

Like other electrophoretic mutation scanning methodologies, one of the primary determinants of sensitivity of CSGE is reputed to be DNA fragment length. In a recent update on CSGE, Ganguly reported a study in which the relative mobilities

Microsatellite Gel Electrophoresis

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.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment