Micromachined pipette arrays.

In this paper, the design and characterization of batch fabricated metallic micromachined pipette arrays is described. The process used to fabricate the micromachined pipette arrays (MPA) includes p+ etch-stop membrane technology, anisotropic etching of silicon in potassium hydroxide, sacrificial thick photoresist micromolding technology, and electrodeposition. Arrays of one to ten pipettes have been fabricated using nickel as the structural material and palladium as the biocompatible coating of inside walls. The inner dimensions of the individual pipettes fabricated to date range from 30 microns to 1.5 mm in width, 0.5 mm to several cm in length, and 5-50 microns in thickness. The center-to-center spacing of these pipettes varies from 100 microns to several centimeters. The MPA have a number of advantages when compared to the current micropipette technology, including the ability to transfer precise volumes of samples in the submicroliter range; the ability to manipulate samples, reagents, or buffers in a highly-parallel fashion by operating hundreds of individual pipettes simultaneously; and the compatibility with the submilimeter center-to-center dimensions of the microscale biochemical analysis systems. The application of the MPA to high lane density slab gel electrophoresis is explored. Sample wells are formed in agarose gels by using micromachined combs (solid MPA) at center-to-center spacing ranging from 250 microns to 1.9 mm. The samples are loaded using the MPA. The results of the micro-gel separations compare favorably with the standard mini-gel separations and show a twofold increase in the number of theoretical plates as well as a sixfold increase in lane density.

Base stacking: pH-mediated on-column sample concentration for capillary DNA sequencing.

An on-column sample concentration method for capillary-based DNA sequencing was studied. This base-stacking method allows direct injection of unpurified products of dye-primer sequencing reactions onto capillaries without any pretreatment. On-column concentration of DNA fragments is achieved simply by electrokinetic injection of hydroxide ions. A neutralization reaction between these OH- ions and the cationic buffer component Tris+ results in a zone of lower conductivity, within which field focusing occurs. DNA fragments are concentrated at the front of this low-conductivity zone. With sample injection times as long as 360 s at 50 V/cm, resolution could still be restored by the stacking process. Using a 36/47-cm-long uncoated capillary, with poly(dimethylacrylamide) as the separation matrix, and electric field of 160 V/cm, a resolution of at least 0.5 could be generated for fragments up to 650 nucleotides long. Both resolution and signal strength are excellent relative to conventional injection of highly purified samples. No significant degradation of the capillary performance was observed over at least 20 sequencing runs using this new sample injection methods.

Fully automated DNA reaction and analysis in a fluidic capillary instrument.

A simple, reliable, automated genetic analysis instrument has been designed and prototyped. The system uses novel fluidic technology, coupling thermal cycling, reaction purification, in-line loading, and capillary electrophoresis in a single instrument. Samples in the loop of an injection valve are amplified inside a rapid air thermal cycler. A liquid chromatographic separation eliminates contaminants and excess salt. The sample is loaded in an efficient, continuous, flow-through manner onto a polymer-filled separation capillary. Detection by laser-induced fluorescence produces signal-to-noise ratios of 1000:1 or greater. Refilling of the polymer-filled capillary is automatic; during the run, the system is reconditioned for injection of another sample. Since all components and connections are fluidic, automation is natural and simple. The instrument is reliable and fast, performing PCR reaction cycling, purification and analysis, all in 20 min. Reproducibility (CV) of retention times is 2% (n = 129) and of peak areas 9% (n = 34). Bubbles and particulates are eliminated by the chromatography column. Adaptation of the instrument prototype for separation of DNA-sequencing reactions is described; cycle sequencing and electrophoresis of a single lane are complete in 90 min. Implications and challenges for development of fully automated fluidic instruments for genomic sequencing are discussed.

Binding of biotinylated DNA to streptavidin-coated polystyrene latex.

The binding of 5'-end biotinylated DNA fragments was compared between streptavidin (SA)-coated commercial M-280 magnetic latex particles with a diameter of 2.8 microns and adsorption-coated polystyrene (PS) latex standard particles whose diameter is 0.272 microns. Amino acid analysis showed the protein content of the commercial particles to correspond to 4x monolayer coverage, while the adsorption-coated PS particles displayed monolayer coverage, or 8 pmol/cm2. A fluorescence-based method was developed to quantify the adsorption of FITC-labeled SA, biotin, and biotinylated DNA. The validity of the method was substantiated for the labeled protein by both amino acid analysis and a colorimetric protein assay. While the specific binding of biotin was 0.38 mol per mole of SA on the adsorption-coated 0.272-microns particles and slightly higher (0.6 mol per mole SA) on the 2.8-microns particles, the specific binding of the bulky biotinylated 300-bp DNA was more favorable on the smaller particle (0.12 mol per mole SA for 0.272 microns versus 0.04 mol per mole SA for 2.8 microns).

Reloading and stability of polyacrylamide slab gels for automated DNA sequencing.

Current automated fluorescent instruments are all based on slab gels that are used once and then discarded. Practitioners of capillary gel electrophoresis often reuse their gels for multiple samples. As slab gels are made thinner to increase speed, the ability to reload new samples after each run will become more desirable. Techniques have previously been developed for reloading and stabilizing capillary gels. The application of these methods to slab gel electrophoresis is reported. Gels are shown to be reusable for at least four consecutive automated runs. The stability of various slab gel formulations and their ability to survive multiple loadings with sequencing samples are compared. Formamide-containing gels are shown to be superior to their urea counterparts. The potential that running buffer additives have for improving automated DNA sequencing is discussed. Residual template DNA in sequencing samples can produce gel instability, reduce resolution and decrease signal. These effects are examined.

Rapid cycle sequencing in an air thermal cycler.

Cycle sequencing using Taq DNA polymerase has gained popularity recently due to reduced template requirements, improved signal and its ability to directly sequence PCR fragments. A major drawback to the technique is the time required for performing reactions in a block-based thermal cycler. To help cycle sequencing compete with other methods, we have modified the protocol to be performed in capillaries using an air-based thermal cycling instrument. This instrument has been developed and optimized for rapid, specific amplification of DNA by PCR. The resulting cycle sequencing methodology is faster than block-based approaches; a reaction can be completed in 25 min, compared with about 2 h in a conventional instrument. Thus, the speed of the technique is competitive with standard uncycled T7 or Taq reactions. Accuracy of the sequencing data is improved; two problem areas in the sequence obtained with a block cycler are ameliorated by the capillary methodology. This technique represents a novel approach to cycle sequencing that will further the development of capillary-based analytical methods.

Stability of capillary gels for automated sequencing of DNA.

Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and immunological characterization of porcine kidney ferredoxin.

Porcine renodoxon is a kidney mitochondrial iron-sulfur protein (ISP) that functions to transfer electron to cytochromes P450 of the vitamin D pathway. A full-length cDNA clone to porcine renodoxin was isolated in the current investigation and used to study the protein's primary structure and immunological properties. The cysteine ligands for the iron-sulfur center, and the surface protein-binding and phosphorylation sites occupied identical positions in both porcine renodoxin and bovine adrenodoxin. Furthermore, porcine renodoxin was functionally indistinguishable from bovine adrenodoxin and the mature forms of both proteins had the same encoded length and shared approximately 91% sequence similarity. A synthetic peptide to the surface protein-binding region was used to demonstrate the antigenicity of the domain in both the porcine and the bovine ISPs. However, porcine renodoxin displayed only limited immunological identity to other regions of bovine adrenodoxin as measured by competitive enzyme-linked immunosorbent assay. Part of this immunological distinction was attributed to the COOH-terminal processing of porcine renodoxin, an action which negated expression of a COOH-terminal antigenic site that is present in bovine adrenodoxin. Other antigenic differences were linked to charged-residue substitutions that were located in predicted surface domains. The highest frequency of surface-residue substitutions in ferredoxin proteins was predicted for porcine renodoxin, which could provide a basis for understanding why the pig protein appears more antigenically divergent than other ferredoxins.

Three DNA sequencing methods using capillary gel electrophoresis and laser-induced fluorescence.

Capillary gel electrophoresis is demonstrated for the four-spectral-channel sequencing technique of Smith, the two-spectral-channel sequencing technique of Prober, and the one-spectral-channel sequencing technique of Richardson and Tabor. Sequencing rates up to 1000 bases/h are obtained at electric field strengths of 465 V/cm. At lower electric field strengths, capillary electrophoresis produces useful data for fragments greater than 550 nucleotides in length with 2 times better resolution than slab gel electrophoresis. An on-column detector produces detection limits of 200 zmol (1 zmol = 10(-21) mol = 600 molecules) for the four-spectral-channel technique. A postcolumn detector, based on the sheath flow cuvette, produces detection limits of 20 and 2 zmol for the two- and one-spectral-channel techniques, respectively.

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.

Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette.

Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported. A post-column laser-induced fluorescence detector based on the sheath flow cuvette was used to minimize background signals due to light scatter from the gel and capillary. A preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments was obtained. The system was used to analyze an actual DNA sequencing sample. Theoretical plate counts of 2 x 10(6) were produced. Gel stability limits the performance of the current system.

Capillary gel electrophoresis for rapid, high resolution DNA sequencing.

Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.

Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity.

Previous work showed that the simple eukaryote Saccharomyces cerevisiae contains a group of RNAs with the general structural properties predicted for small nuclear RNAs (snRNAs), including possession of the characteristic trimethylguanosine 5'-terminal cap. It was also demonstrated that, unlike their metazoan counterparts, the yeast snRNAs are present in low abundance (200-500 molecules per haploid cell). We have now used antibody directed against the 5' cap to investigate the total set size of snRNAs in this organism. We present evidence that the number of distinct yeast snRNAs is on the order of several dozen, that the length of the capped RNAs can exceed 1000 nucleotides, and that the relative abundance of a subset of these RNAs is 1/5th to 1/20th that of the class of snRNAs described previously. These findings suggest that the six highly abundant species of snRNAs (U1-U6) typically reported in metazoans may represent a serious underestimation of the total diversity of snRNAs in eukaryotes.

The Encircler. A new instrument for the performance of the Thiersch procedure for rectal procidentia.

A new instrument for the performance of the Thiersch procedure is presented. It offers advantages over the traditional method of doing this operation in that it can be done through only one incision, reducing both the risk of infection and the trauma to perianal tissues.

Structure of intron-containing tRNA precursors. Analysis of solution conformation using chemical and enzymatic probes.

Using chemical and enzymatic structure-specific probes adapted to rapid gel sequencing techniques, we have analyzed the solution conformations of precursors to two yeast tRNAs which contain an intervening sequence, pre-tRNAPhe and pre-tRNATyr. Interpretation of the data was greatly facilitated by performing direct mature/precursor tRNA comparisons. In addition, the effects of tertiary interactions on probe specificity could be evaluated from the results obtained with mature tRNAPhe, whose crystal structure is known. We find: 1) the folding of the precursor CCA terminus, acceptor stem, T psi C stem, variable loop, anticodon stem, and D stem identical with that of the equivalent regions in the cognate, mature tRNA. 2) The T psi C loop and D loop appear to vary slightly in tertiary structure between mature and precursor species. 3) The precursors contain a helix involving the anticodon triplet and a complementary sequence in the intron. 4) The stability of this helix is much greater for pre-tRNAPhe than for pre-tRNATyr. 5) The splice sites for both precursors are located in single-stranded loops. These results bear out predictions based on genetic analyses and are consistent with the view that recognition of universally conserved features of tRNA structure allows all tRNA precursors containing intervening sequences to be processed by a single splicing apparatus.

Yeast contains small nuclear RNAs encoded by single copy genes.

We have identified a group of RNA molecules in Saccharomyces cerevisiae that appears to be equivalent to the U class of small nuclear RNAs previously described in other eucaryotes, resembling them in size, metabolic stability, 5' cap structure, presence of modified bases, and nuclear localization. However, the yeast snRNAs differ from their counterparts in several potentially important ways. First, they are present in very low abundance, less than 200 copies per cell, as compared to 10(5)-10(6) for mammalian U1-U6. Second, there appear to be more species in yeast than in any cell type previously examined. Finally, we have cloned five yeast snRNA genes, and find that each is present in a single copy per haploid genome, whereas all previously characterized snRNAs are encoded by multiple (5 to 100) gene copies. The presence of single copy genes in yeast will greatly facilitate the genetic analysis of snRNA function.