Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.

The use of fluorescence detection and internal lane standards to size PCR products automatically.

We have developed chemical procedures, optical and electrophoretic instrumentation and computer software automate the analysis of polymerase chain reaction (PCR) products. DNA molecules labeled with up to four different fluorescent dyes are analyzed within a single electrophoresis gel lane. A size calibration curve is created for each electrophoresis lane from the electrophoretogram of uniquely labeled DNA fragments belonging to an internal lane standard that co-electrophoreses with the PCR products. The unknown molecular lengths of PCR products are automatically calculated from the calibration curve. Data from control experiments with DNA segments of known molecular length demonstrate the accuracy and precision of such sizing. This system has been applied to the analysis of PCR products for research in the areas of human identification, genetic mapping and genetic disease.

Automated genetic analysis.

Automation of several new, non-traditional techniques for genetic analysis has now become possible. A new system is described that performs gel electrophoretic analysis of DNA including VNTRs, gene segments, and restriction enzyme digests. The instrument detects emitted fluorescence from labeled DNA segments in real-time as they electrophore through a gel matrix past a scanning laser beam. Molecular length determination and band quantification is accomplished by comparison to an in-lane standard. Since DNA segments can be labeled and detected with any of four different dyes, the simultaneous analysis of similar length segments from different reactions within a single lane is possible. PCR products are analyzed for research in the areas of human identification and genetic disease. These examples illustrate how automation will play key role in this new era of genetic analysis.

Automation of specific human gene detection.

An instrument/chemistry system is described that automates a new chemical procedure functionally equivalent to Southern blotting. A fluorescence gel scanner that detects migrating DNA fragments in real-time analyzes the samples produced by a prototype liquid-handling instrument that automates a solution-phase hybridization/solid-phase capture chemistry for DNA analysis. The combination of this chemistry, the gel scanner, and robotic automation eliminates the tedium encountered in traditional manual methods for specific gene detection and reduces analysis time from days to hours. Restriction fragment lengths are measured with high precision by comparison with in-lane standards to minimize effects attributable to migration anomalies. The utility of this automated system is demonstrated by executing a clinical research application involving hybridization to a multi-copy repeat sequence on the Y chromosome and its detection.

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition.

A significantly improved method for base composition analysis of synthetic oligodeoxyribonucleotides is presented. This highly accurate and sensitive method used enzymatic digestion followed by high-resolution HPLC of the nucleosides to determine the empirical base composition of the parent compound. The enzymatic digestion reaction is quantitative and is not blocked by modified bases, thus allowing the degree of base deprotection and chemical modification to be assessed. Digestion data are presented for oligodeoxyribonucleotides which range from 18 to 150 bases in length with excellent agreement of experimental and theoretical composition. The method is also applicable to high-molecular-weight genomic DNA.