News
FROM THE ACS NATIONAL MEETING IN ORLANDO
Applying laser-induced fluorescence to biotechnology Slab-gel electrophoretic DNA sequencing is a laborious method that can only sequence fragments 800 to 1200 bases, or ~ 1 kb, long. Imagine a method that can sequence fragments tens of kb long. Richard Keller and colleagues at Los Alamos National Laboratory are developing a laser-induced fluorescence (LIF) approach that has the potential to sequence such fragments at a rate of 100-1000 bases per second. Keller and coworkers have been able to detect single fluorescently labeled nucleotides cleaved from DNA fragments, but they have not yet demonstrated actual sequencing. A single molecule passing through a focused laser beam emits a burst of ~ 10,000 photons; ~ 1% of these photons are collected and detected. A burst of ~ 100 photoelectrons on top of a random background is the signature of a single molecule. For the most efficient detection, the molecule must pass through the center of the laser beam. A threshold in the burst size can be set to detect most of the molecules while making very few falsepositive identifications. In addition to DNA sequencing, the LIF detection method can be used for DNA fragment sizing, which is important for disease diagnosis and personnel identification. DNA fragments are stained with a fluorescent intercalating dye. The sample is diluted, and stained fragments are passed individually through a focused laser beam. The fluorescence intensity from each fragment is directly proportional to the amount of dye that it intercalates, which in turn is directly proportional to the size of the fragment. Keller's group uses the dyes TOTO and YOYO, neither of which fluoresce significantly when 658 A
A family reunion, of sorts. Five generations of active analytical chemists gathered at the symposium honoring Norman Dovichi at the ACS meeting. Arranged (L-R) youngest to oldest, with advisor standing to the right of advisee, they are Darryl Bornhop of Texas Tech University, Dovichi of the University of Alberta, Joel Harris of the University of Utah, Fred Lytle of Purdue University, and David Hercules of Vanderbilt University.
alone but which fluoresce intensely when intercalated in DNA. In comparison with gel electrophoresis, sizing DNA fragments by this approach is much faster, requires less DNA, and is independent of DNA conformation. LIF is particularly suited for sizing large fragments (> 50 kb, where pulsed-field gel electrophoresis must be used), but it does not work as well as gel electrophoresis for sizing small fragments. The smallest fragment that Keller's group has been able to size is 212 kb.
Itsy-bitsy, teeny-weeny beakers We've probably all seen small beakers at one time or another, but picoliter beakers? Rose Clark, a postdoctoral associate of Andrew Ewing at Pennsylvania State University, described how polystyrene picoliter "beakers" can be made from templates that are etched into silicon wafers and used for electrochemical experiments. The vials allow analysis of single cells without the dilution required in traditional analyses. The beakers were made with lithographically fabricated templates that imprint the vials in polystyrene. The resulting beakers hold 20 pL and can handle volumes as small as 1 pL. Ewing and
Analytical Chemistry News & Features, November 1, 1996
Clark have studied the electrochemical response of catecholamine from single adrenal cells in the beakers and found that cyclic voltammograms of the system showed no distortion. Single adrenal cells were isolated by culturing the cells on microvial substrates and then using pressure injection with a "picospritzer".
Detecting a single-base genetic mismatch with CE Rapid and accurate analysis of genetic variations is vital for diagnosis of genetic diseases. Maria Dulay of Richard Zare's group at Stanford University investigated using high-temperature free-solution CE to detect a single base genetic sequence mismatch in the gene linked to cystic fibrosis. They used a hybridization mixture containing the target gene and its peptide nucleic acid (PNA) analogue, a fully synthetic DNArecognizing ligand with a neutral peptidelike backbone and purine and pyridinebased nucleobases, and showed that the PNA probe only hybridizes with the DNA sequence that is a complementary perfect match. Even a single base mismatch cannot hybridize with the PNA probe at the hightemperature experimental conditions. The hybridization and separation are completed in less than 15-20 min.
