A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with greater than 99% accuracy.

Short tandem repeat (STR) analysis is increasingly being used in forensic case analysis because of the large number of STR loci in the human genome and their highly polymorphic nature. An automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR allele patterns from simulated forensic samples. The amplification strategy used a 19 base pair extension on the 5′ end of one of the PCR primers. This sequence is identical to the sequence of a universal M13 Forward sequencing primer which is included in the amplification reaction. Allelic bands were detected by incorporation of the M13 primer-fluorescent dye conjugate into PCR products thus eliminating the need for direct conjugation of fluorescent dye to individual STR primers. By using an IR-based automated DNA sequencer and Tth DNA polymerase, polymorphic STR alleles were detected on-line rapidly and efficiently from bloodstains using only a high temperature incubation to extract DNA from blood cells. Five STR loci were also amplified using Chelex extracted DNA from simulated forensic samples. Multiplexing of three primer pairs in a single PCR mixture for amplification was accomplished using Taq polymerase. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for radioactivity and the gel handling required with silver staining and fluor detection systems. Real-time detection permits immediate visualization of the data and STR alleles are displayed as familiar autoradiogramlike images that can be analyzed by computer. By loading a 64 lane gel twice and multiplexing with three primer pairs, forensic scientists can type at least three loci from 120 samples in one day.

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.

SUMMARYMutants of cauliflower mosaic virus (CaMV), generated in vitro by modification of recombinant DNA plasmids containing the viral genome, either retained the ability to induce disease symptoms on turnip plants, produced less severe symptoms or failed to induce symptoms. Wild-type symptoms were produced by a variant CaMV DNA of the Cabbage S isolate that had 4 bp in open reading frame (ORF) III replaced with a 16 bp sequence. Less severe symptoms, due to a delay in symptom appearance relative to inoculation with wild-type DNA, were induced by a mutant with a frameshift mutation in ORF II (pSA103). CaMV DNA, recovered from plants infected with pSA103, contained a second mutation which restored the original translation reading frame. Nucleic acid hybridization to 'squishes' of leaf tissue from plants that had been inoculated with mutant DNAs that included DNAs modified in each of the six major ORFs of CaMV DNA revealed that only those plants that appeared diseased had detectable CaMV nucleic acid in uninoculated leaves. Replicated CaMV DNA was also not detected in non-encapsidated and virion DNA fractions from inoculated leaves of non-diseased plants.

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.