Lincoln Mcbride

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.

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.

Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis

Abstract Improvement of the hydrogen-phosphonate method for oligodeoxynucleotide synthesis has been achieved via an efficient capping reagent, triethylammonium isopropylphosphite, resulting in stable 5′ isopropylphosphate failure sequences. Activation for capping and coupling is best effected with 1-adamantanecarbonyl chloride.

Base Modification and the Phosphoramidite Approach

Abstract Oligodeoxynucleotides 18–150 bases in length were synthesized with both 0-(2-cyanoethyl)- and 0-(methyl)-phosphoramidites. After enzymatic degradation of the purified products, base modification and composition were evaluated by HPLC. Additionally, synthesis of 5′-d[GCGCGCTT] with O-(methyl) phosphorus protection generated 3-methylthymidine when thiophenoxide was omitted from the deprotection protocol.

Quantitative PCR Technology

Higuchi et al. (1992, 1993) pioneered the analysis of PCR kinetics by constructing a system that detects PCR products as they accumulate. This “real-time” system includes the intercalator ethidium bromide in each amplification reaction, an adapted thermal cycler to irradiate the samples with ultraviolet light, and detection of the resulting fluorescence with a computer-controlled cooled CCD camera. Amplification produces increasing amounts of double-stranded DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence versus cycle number, the system produces amplification plots that provide a more complete picture of the PCR process than assaying product accumulation after a fixed number of cycles.