David L. Steffens

An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase–DNA complexes to surfaces

Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9°N in which two biotinylated peptide ‘legs’ are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase–DNA–streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase–DNA–streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis.

A Near-Infrared, Surface-Enhanced, Fluorophore-Linked Immunosorbent Assay

The enzyme-linked immunosorbent assay is commonly used for research and clinical applications but typically suffers from a limited linear range and is difficult to multiplex. The fluorophore-linked immunosorbent assay is a closely related technique with good linear range and the ability to detect multiple antigens simultaneously but is typically less sensitive. Here, we demonstrate a near-infrared, surface-enhanced fluorophore-linked immunosorbent assay with sensitivity comparable to its enzyme-linked counterpart. A 59-fold enhancement to sensitivity (slope of linear fit) and an 8-fold improvement in LOD are demonstrated on a direct assay with rabbit immunoglobulin-G as a model system. The technique is also tested on a clinically relevant assay to detect alpha-fetoprotein, in which a 42-fold enhancement to sensitivity is demonstrated along with a 16-fold improvement in LOD. The technique enables these accomplishments while maintaining the entire traditional assay protocol and simply adding two steps at the end. This technique may prove superior to current protocols for biomarker research and clinical diagnoses, which require high sensitivity along with quantitation over an extended range.

Multiplex amplification of STR loci with gender alleles using infrared fluorescence detection.

The analysis of short tandem repeat (STR) polymorphisms has proven extremely useful for gene mapping, paternity testing, and forensic analysis. Several commercial products are currently available for performing amplification and analysis of STRs. We have adapted Promega Geneprint Systems for use with a high sensitivity infrared (IR) fluorescent automated DNA sequencer. IR-labeled amplification products are generated by including a small quantity of IR-labeled dATP in the reaction. Several Geneprint STR loci can be multiplexed together with the amelogenin sex identification locus in a single amplification reaction. We have successfully amplified up to five Geneprint STR loci together with the amelogenin locus thus improving the throughput of analysis. Purified genomic DNA as well as simulated forensic samples have been utilized for these multiplex amplifications.

STR Typing without DNA Extraction Using an Infrared-Based Non-Radioactive Automated DNA Sequencer

A LI-COR Model 4000 automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR allele patterns from bloodstains and simulated forensic samples using Tth polymerase. Two different amplification strategies were used for labeling. Multiplexing of three primer pairs in a single PCR amplification was accomplished using Taq polymerase. Typing of STR alleles was also achieved using a GenePrint™ STR System (Promega) for various forensic-like specimens. Genotyping of the O allele of the human ABO blood group locus as well as the gender differentiating amelogenin locus was also performed using both strategies. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for radioactivity and the gel handling required with some methodologies. STR alleles are displayed as autoradiogram-like images during the run and can be computer analyzed.