Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2'-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis.

Standards and controls in DNA assays.

Automated methods are described that begin to standardize genetic analysis at the DNA level. These new methods complement existing automation such as DNA extraction, liquid-handling robotics, thermal cycling and computer information management systems. Using electrophoresis, several independent genetic loci can be analyzed simultaneously using multi-color fluorescent gel scanning detection where DNA segments labeled with up to four different fluorescent dyes are analyzed within a single electrophoresis lane. The amounts and molecular lengths of PCR products are determined with high accuracy and precision by co-electrophoresis with a uniquely colored internal standard. These new methods have the potential of minimizing variations in results among different laboratories.

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.

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.

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.

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

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.