Showing papers by "Applied Biosystems published in 1993"

Allelic discrimination by nick-translation PCR with fluorogenic probes.

Abstract: Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry

Abstract: Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms

Abstract: Pregnant women over 35 years of age are routinely offered screening tests and karyotyping to detect Down syndrome and certain other aneuploidies because the risk of these disorders increases exponentially with maternal age It is, however, only cost-effective to karyotype high-risk pregnancies and a substantial number of remaining aneuploidy cases go undetected We describe a rapid, inexpensive method to detect trisomy using polymorphic small tandem repeat (STR) markers and the polymerase chain reaction (PCR) to amplify amniocyte DNA STR patterns obtained on patients with trisomy 21, trisomy 18 and triplo-X syndromes are distinct from controls Polymorphic trisomy genotypes either show three fragment peaks of equal intensity or two fragments at a 2:1 dosage ratio In addition, Turner syndrome (45, X0) DNA can be distinguished from normal male DNA because it fails to amplify a Y-chromosome specific PCR marker yet contains only a single dose of X-specific STR markers Quantitative analysis of peak heights and areas from STR markers show that the two peak patterns separate into completely non-overlapping groups The high level of heterozygosity of most STR markers result in a predominance of heterozygous controls and trisomy patterns with multiple alleles, the easiest patterns to differentiate Homozygosity, and hence an uninformative STR pattern, is more common in controls than in trisomy samples We anticipate as few as three STR markers per chromosome should be over 99% informative

Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing.

Abstract: A comparison has been made between manual and automated DNA sequencing procedures to evaluate the ability to distinguish mixtures of wild-type and mutant sequences. Quantitative detection of such mixtures of HIV-1 drug resistance mutations was best achieved using an automated system that uses fluorescent-labelled sequencing primers. This procedure has a wide range of applications in clinical research, including heterozygote analysis. Software that automatically reports mixed-base positions is presented.

Structural heterogeneity in HLA‐B70, a high‐frequency antigen of black populations *

Abstract: Although the B70 antigen exhibits allele frequencies of 8-23% in African and American black populations, it remains poorly defined. Cloning and sequencing of cDNA encoding B70 antigens from six cell lines has identified a group of three closely related alleles: B*1503, B*1509 and B*1510, that form a subgroup of the B15 family. The sequences of these alleles and, in particular, B*1503, are close to that of the HLA-B consensus consistent with the difficulty in their serological definition. The products of the three alleles correspond to three electrophoretically detected variants of the B70 antigen and some correlation with the B71 and B72 subspecificities of the B70 antigen can be made. A fourth allele, B*7901, previously described by Choo et al. (J. Immunol. 147: 174-180, 1991) that was not serologically typed as B70, differs by a single nucleotide substitution from B*1510. The sensitivity of alloantibodies to single differences in peptide binding residues suggest a role for bound peptides in the HLA-B70 alloantigenic specificities. The heavy chains encoded by the four alleles differ at four peptide binding residues of the antigen recognition site, the evolutionary modification of which can be explained in terms of interallelic recombination events.

Viscous electrophoresis polymer medium and method

Abstract: A viscous electrophoresis separation medium is disclosed. The medium is formed by a matrix formed by a copolymer composed of hydrophilic polymer segments having selected, substantially uniform segment lengths, and a plurality of hydrophobic polymer segments carried on, and spaced from one another by the hydrophilic polymer segments. Also disclosed is an electrophoresis method which employs the separation medium, and novel copolymers used in forming the medium.

Synthesis and characterization of 5'-fluorescent-dye-labeled oligonucleotides.

Abstract: Fluorescent-dye-labeled oligonucleotides are used in many procedures, including DNA sequencing, PCR, restriction mapping, the study of genetic disease, and forensics identification. In this paper, we describe detailed methods for the synthesis, purification, and quantification of 5'-fluorescent-dye-labeled oligonucleotides. The relationship of specific and nonspecific dye attachment to synthetic oligonucleotides is discussed, and the importance of primer design is considered as a way to prevent chemical problems from interfering with biological reactions.

Capillary electrophoresis molecular weight separation of biomolecules using a polymer-containing solution

Abstract: A capillary tube with a charged inner surface for use in capillary electrophoresis and methods. The tube is filled with an electrolyte solution containing 0.05-30 % weight/weight of a hydrophilic polymer which is characterized by (A) a molecular weight of 20-5,000 kilodaltons, and (B) a charge between 0.01 and 1.0 % as measured by the molar percent of charged monomer subunits to the total polymer subunits, where said charged monomer subunits have the charge opposite to the wall charge at a selected electrophoresis pH.

Isolation of a putative fimbrial adhesin from Bordetella pertussis and the identification of its gene.

Abstract: Summary We report the purification of a minor Bordetella pertussis fimbrial subunit, designated FimD, and the identification of its gene (fimD.) FimD could be purified from the bulk of major fimbrial subunits by exploiting the fact that major subunit-subunit interactions are more stable in the presence of SDS than minor-major subunit interactions. To locate the gene for FimD, internal peptides of FimD were generated, purified and sequenced. Subsequently, an oligonucleotide probe, based on the primary sequence of one peptide, was used to clone fimD. The primary structure of FimD, derived from the DNA sequence of its gene, showed homology with a number of fimbrial adhesins. Most pronounced homology was observed with MrkD, a fimbrial adhesin derived from Klebsieila pneumoniae. These observations suggest that FimD may represent a B. pertussis fimbrial adhesin. With a fimD-specific probe we detected the presence of a fimD homologue in Bordetella parapertussis and Bordetella bron-chiseptica but not in Bordetella avium. Cloning and sequencing revealed that the B. parapertussis and B. bronchiseptica fimD product differed from the B. pertussis fimD product in 20 and 1 amino acid residues, respectively. Since B. bronchiseptica is normally not a human pathogen, but causes respiratory disease in a wide range of non-human mammalian species, this may suggest that FimD recognizes a receptor that is well conserved in mammalian species. An in-frame deletion in fimD completely abolished FimD expression and also affected the expression of the major subunits Fim2 and Fim3 suggesting that, in contrast to other adhesins that are minor components of fimbriae, FimD is required for formation of the fimbrial structure.

Method and probe composition for detecting multiple sequences in a single assay

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In one embodiment of the invention, a plurality of different-sequence probe pairs are added to a target polynucleotide, where each probe pair includes two polynucleotide probe elements which are complementary in sequence to adjacent portions of a selected one of the target sequences in the target polynucleotide. In each probe pair, one of the probe elements contains a non-polynucleotide polymer chain which imparts a distinctive mobility to the associated probe pair, when the elements in the pair are ligated. The other element in the pair contains a detectable reporter label. After the probe pairs have been allowed to hybridize with the target polynucleotide, the hybridized polynucleotides are treated under conditions effective to ligate the end subunits of target-bound probe elements when their end subunits are base-paired with adjacent target bases. The ligated probe pairs are then released from the target polynucleotide and separated electrophoretically in a sieving matrix, or chromatographically.

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Abstract: Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner™ in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc.

Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix.

Abstract: Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge-to-mass ratios for SDS-glycoprotein complexes, resulting in over-estimation of molecular weights by SDS-PAGE. In order to minimize these inaccuracies for proteins with abnormal charge-to-mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on-line monitoring which allows quantitation and precludes post-separation staining and destaining of gels.

High efficiency fluorescence flow cell for capillary liquid chromatography or capillary electrophoresis

Abstract: A fluorescence flow cell having a capillary through which a sample fluid is moved, an optical source that directs an exposing beam of light through the sample fluid, a collector that collects fluorescent light from the exposed region of the sample and directs such collected light to a detector. A beam blocker prevents light that is scattered by the capillary walls from reaching the detector, thereby improving the signal-to-noise ratio of the system. The beam blocker also has a pair of legs that prevent the sample fluid from being exposed by either the exposing light or the collected light outside of the exposing region. The structure of the components enables the various components to be quickly and accurately assembled and enables easy replacement of the capillary.

Sequence of mouse glucose-6-phosphate dehydrogenase cDNA.

Abstract: A full-length mouse glucose-6-phosphate dehydrogenase (G6PD) cDNA has been isolated and sequenced, and the evolutionary conservation of many portions of the sequence has been verified by comparison with that of human and other sources.

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1a by multicolor in situ hybridization

Abstract: Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) result in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. the smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%). © 1993 Wiley-Liss, Inc.

Alternative labeling techniques for automated fluorescence based analysis of PCR products.

Abstract: We present here convenient and simple methods that utilize two new fluorescent tags, TOTO-1 and fluorescein-12-dUTP, in conjunction with analysis of PCR products on automated, fluorescence-based electrophoretic instruments. These methods should prove valuable in those applications wherein the effort or expense of covalently attaching a fluorescent tag onto one or both of the PCR primers is not justified. Advantages and disadvantages of the new labeling methods are then reviewed.

Micellar capillary electrophoresis of the ecdysteroids

Abstract: The technique of micellar capillary electrophoresis has been applied to the separation of ecdysteroids (polyhydroxylated insect hormones) both as pure standards and in the extracts of plants and insect eggs. Succesful separations of a range of closely related ecdysteroids were obtained, however, the quality of the result was found to be critically dependent on the solvent in which the sample was applied and the degree of purity of the extracts. The technique was found to be suitable for the analysis of purified ecdysteroid-rich extracts of plants (Silene nutans, S. otites) and the eggs of the desert locust (Schistocerca gregaria). The elution order of the analytes was similar to that obtained using reversed-phase HPLC.

Methods and compositions for detecting base pair mismatches

Abstract: The present invention discloses improved compositions and methods for detecting mutations, including single base changes, in nucleic acid sequences using RNase protection assays. The improvements include concomitant, dramatic reductions in the salt and RNase enzyme concentrations in the RNase digestion reaction mixture which result in greater sensitivity in detecting genetic mutations. Another embodiment of the present invention is kits to be used for the detection of single base mismatches in nucleic acid samples.

Method of forming N-protected amino acid thiohydantoins

Abstract: A method of forming a thiohydantoin of formula (II) from an N-protected amino acid (I) is described. The method employs a uronium (III) or phosphonium (IV) compound to activate the terminal carboxyl group of the amino acid and a thiocyanate reagent to cyclize the activated amino acid to the thiohydantoin. The thiohydantoin can be cleaved from its N-protecting group, for use in C-terminal peptide sequencing. Particularly preferred uronium compounds include salts of 2-chlorouronium. Preferred thiocyanate reagents include trimethylsilyl isothiocyanate and crown ether adducts of metallothiocyanates, such as the 18-crown-6 adduct of KSCN.

Trityl monitoring of automated DNA synthesizer operation by conductivity : a new method of real-time analysis

Abstract: AutoAnalysis is a new method for detecting and quantitating the trityl cation released each cycle on automated DNA synthesizers. The trityl (dimethoxytrityl) cation is removed from the growing oligonucleotide after each base addition and is a useful measure of synthesis efficiency. The traditional absorbance method of collecting each trityl effluent with a fraction collector, followed by dilution with an acid solution and careful quantitation by UV/VIS spectroscopy is costly, tedious and prone to error. The absorbance method for trityl cation analysis must usually wait until the synthesis is complete. Interruption of a failed operation, for a variety of reasons, such as an empty reagent reservoir, is thus not possible. Taking advantage of the conductive properties of the trityl cation, immediate and real-time quantitation is now possible by integrating the total conductance of the flowing stream during the detritylation step after each nucleoside addition in DNA synthesis. A conductivity cell is mounted downstream, past the synthesis column. The conductivity signal is processed and displayed as the current average stepwise yield and overall yield. If the yield drops below a pre-set threshold value because of a failure situation, the synthesizer will interrupt, preserving reagents. AutoAnalysis allows trityl monitoring with complete automation on the Applied Biosystems Models 392 and 394 DNA/RNA Synthesizers.

Large Scale Synthesis of Oligonucleotides via Phosphoramidite Nucleosides and a High‐Loaded Polystyrene Support.

Abstract: Large scale quantities of phosphodiester and phosphorothioate oligonucleotides are synthesized on an aminopolyethyleneglycol derivatized polystyrene (TentaGel) support. Efficient, automated synthesis up to 1 mmole scale is achieved with phosphoramidite nucleoside monomers and 5-ethylthiotetrazole activator.

Probe method and composition for detecting different dna sequences

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In practicing the method, a plurality of different-sequence probe pairs are added to a target polynucleotide, where each probe pair includes two polynucleotide probe elements which are complementary in sequence to adjacent portions of a selected one of the target sequences in the target polynucleotide. In each probe pair, one of the probe elements contains a non-polynucleotide polymer chain which imparts a distinctive elctrophoretic mobility in a sieving matrix, to the associated probe pair, when the elements in the pair are ligated. The other element in the pair contains a detectable reporter label. After the probe pairs have been allowed to hybridize with the target polynucleotide, the hybridized polynucleotides are treated under conditions effective to ligate the end subunits of target-bound probe elements when their end subunits are base-paired with adjacent target bases. The ligated probe pairs are then released from the target polynucleotide and separated electrophoretically in a sieving matrix, or chromatographically.

High Resolution Full-Range (pI = 2.5 to 10.0) Isoelectric Focusing of Proteins and Peptides in Capillary Electrophoresis

Abstract: Publisher Summary This chapter discusses high-resolution full-range isoelectric focusing (IEF) of proteins and peptides in capillary electrophoresis (CE). IEF is a method that separates proteins on the basis of their isoelectric points (PI). It is accomplished by the electrophoresis of proteins or peptides through a stable pH gradient until their net charge is zero. At this pH, mobility is zero, and the isoelectric point is achieved. This method has been widely used in research, pharmaceutical, and clinical areas to separate, isolate, and analyze proteins from various biological products. The chapter discusses the effects of sample buffer matrix, surfactants, denaturant, and narrow pH range ampholyte using the same CE-IEF method. The study indicated that the CE-IEF method developed by Chen and Wiktorowicz has proven to be very flexible. This CE-IEF method used on a free solution separation can be used to perform rapid high-resolution analysis of proteins and peptides with various sample buffer matrices and additives.