WAVE®/WAVE-HS Nucleic Acid Fragment Analysis System Application Notes

A series of Application Notes have been developed that describe methods for performing various molecular biology applications on Transgenomic Inc.'s WAVE®/WAVE-HS Nucleic Acid Fragment Analysis System. A brief description of each Application Note can be found below. After reading the descriptions, if you would like to view any of the Application Notes in their entirety, please visit our new website (http://www.transgenomic.com/), where you can download the complete publication.

Application Note 101: "Detection of Mutations and Polymorphisms on the WAVE® Nucleic Acid Fragment Analysis System". Describes an automated, rapid, high-throughput, and reproducible scanning method for mutation detection and determination of polymorphisms. Mutations appear as a characteristic pattern of peaks corresponding to the mixture of DNA heteroduplexes and homoduplexes formed when wild type and mutant DNA are hybridized together. Four key aspects of mutation detection are analyzed: PCR primer design, PCR protocol, separation gradient, and separation temperature.

Application Note 102: "Separation and Purification of Short Tandem Repeat (STR) DNA Fragments Using Temperature Modulated Heteroduplex Analysis". Describes the analysis of STRs by a rapid and reproducible method called denaturing HPLC (dHPLC) or Temperature Modulated Heteroduplex Analysis (TMHA), which employs finely tuned partial denaturing temperature gradients together with Transgenomic's DNASep technology to optimize the separation of STR hetero- and homoduplexes. Detailed protocol about separation and purification of F13A locus alleles on human chromosome 6 is presented. Automated sequencing is performed directly from the purified alleles without any additional preparative steps, thus offering dramatic savings in both time and labor.

Application Note 103: "Quality Control and Purification of Oligonucleotides on the WAVE® Nucleic Acid Fragment Analysis System". Describes the high performance capabilities for quality control of oligonucleotide synthesis and oligonucleotide preparative purification. The methods described are based on size dependent separations for performing failure sequence analysis; and size and sequence dependent separations for purification and analysis. The WAVE® System enables automated, high-throughput screening of oligonucleotide synthesis fidelity.

Application Note 104: "PCR Amplification and Automated Sequencing of DNA Fragments Isolated with the WAVE System". DNA fragments isolated on the WAVE® Nucleic Acid Fragment Analysis Platform are shown to be suitable for use in PCR amplification and automated sequencing. This makes possible the use of this technology for preparation of fragments for applications such as reamplification of DNA obtained with random primers, with high cycle number PCR reactions or with nested primer sets and for the subsequent analysis of mutations or polymorphisms identified by screening using TMHA.

Application Note 105: "Competitive Quantitative PCR on the WAVE® Nucleic Acid Fragment Analysis System". Describes a precise, high-throughput method for gene quantitation in microscopic tissue samples. Peak areas corresponding to the heteroduplex species formed by the target and the competitor cDNA, the target, and the competitor are measured accurately by the WAVE® System software. The data are then used to calculate the gene expression levels.

Application Note 106: "Automated and Rapid Genotyping of Mouse Colonies with the WAVE® Nucleic Acid Fragment Analysis System". Describes a rapid and gel-free method for genotyping of mouse mutations using the WAVE® Nucleic Acid Fragment Analysis System and DNASep technology. This approach also permits automated genotyping of transgenic mice, notably by providing an accurate, reliable method for resolving PCR products with subtle differences in base pair lengths in less than 7 minutes.

Application Note 107: "Sizing of DNA Fragments with the WAVE® Nucleic Acid Fragment Analysis System". Describes the DNA sizing methods and buffer gradients used for analysis of plasmid digests and PCR products. The analysis of dsDNA fragments with length 80 to 458 bp demonstrates that retention times of fragments are highly reproducible with standard errors of the mean within the range of 0.0 to 0.03.

Application Note 108: "Detection of Mutations in the ATM Gene: Comparison of Temperature Modulated Heteroduplex Analysis (TMHA) with Single Strand Conformation Polymorphism (SSCP)". Detection of mutations in the gene ATM, responsible for Ataxia-Telangiectasia, was performed using SSCP and TMHA on the WAVE® Nucleic Acid Fragment Analysis System. A comparison between the two methods revealed that TMHA was effective in elucidating mutations in exon 42 in breast cancer patients, while various SSCP optimization protocols failed to identify these mutations.

Application Note 109: "Enhancing Core Facility Services with the WAVE Nucleic Acid Analysis System Technology". Examination of how the capabilities of WAVE Nucleic Acid Fragment Analysis System can expand the services offered by a core facility. Principal application areas include: mutation and polymorphism discovery and screening and rapid genotyping. In addition, the WAVE® system can be used for preparative applications like oligonucleotide purification and quality control, plus preparation of fragments for differential display, sequencing or cloning.

Application Note 110: "Microsatellite DNA Analysis with the WAVE® Nucleic Acid Fragment Analysis System". Describes the use of WAVE® Nucleic Acid Fragment Analysis system for the separation, sizing and typing of alleles from the STR locus HUMTH01 (intron 1 of human tyrosine gene, chromosome position 11p15-15.5). Internal sizing standards were used and the precision of the method was analyzed.