Showing papers by "Applied Biosystems published in 1997"
TL;DR: SSR technology presents the potential advantages of reliability, reproducibility, discrimination, standardization and cost effectiveness over RFLPs, and represents the optimum approach for the identification and pedigree validation of maize genotypes compared to other currently available methods.
Abstract: The utility of 131 simple sequence repeat (SSR) loci to characterize and identify maize inbred lines, validate pedigree, and show associations among inbred lines was evaluated using a set of 58 inbred lines and four hybrids. Thirteen sets of inbred parent-progeny triplet pedigrees together with four hybrids and their parental lines were used to quantify incidences of scoring that departed from expectations based upon simple Mendelian inheritance. Results were compared to those obtained using 80 restriction fragment length polymorphism (RFLP) probes. Over all inbred triplets, 2.2% of SSRs and 3.6% of RFLP loci resulted in profiles that were scored as having segregated in a non-Mendelian fashion. Polymorphic index content (PIC, a measure of discrimination ability) values ranged from 0.06 to 0.91 for SSRs and from 0.10 to 0.84 for RFLPs. Mean values for PIC for SSRs and RFLPs were similar, approximately 0.62. However, PIC values for nine SSRs exceeded the maximum PIC for RFLPs. Di-repeats gave the highest mean PIC scores for SSRs but this class of repeats can result in “stutter” bands that complicate accurate genotyping. Associations among inbreds were similar for SSR and RFLP data, closely approximating expectations from known pedigrees. SSR technology presents the potential advantages of reliability, reproducibility, discrimination, standardization and cost effectiveness over RFLPs. SSR profiles can be readily interpreted in terms of alleles at mapped loci across a broad range of maize germ plasm. Consequently, SSRs represent the optimum approach for the identification and pedigree validation of maize genotypes compared to other currently available methods.
877 citations
TL;DR: The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.
Abstract: We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.
317 citations
TL;DR: A set of four energy transfer dyes are synthesized and demonstrated their use in automated DNA sequencing and their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.
Abstract: We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.
197 citations
TL;DR: The TaqMan LS-50B PCR Detection System facilitates the automated and direct detection of polymerase chain reaction (PCR) products and a modified sample preparation protocol using the EnviroAmp kit was chosen for subsequent studies because it was reliable, easy to use and efficient for the isolation of PCR-amplifiable DNA from foods.
191 citations
TL;DR: Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles.
Abstract: Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b6f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.
172 citations
TL;DR: The ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures is demonstrated, resulting in equivalent performance for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes.
Abstract: We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.
159 citations
TL;DR: It is shown that gene loss after duplication of the prototypical vertebrate Hox clusters is a key feature of both tetrapod and fish evolution.
Abstract: Organization of the Fugu rubripes Hox clusters: evidence for continuing evolution of vertebrate Hox complexes
144 citations
TL;DR: A rapid and efficient non-organic method for extraction of DNA from semen is described, and the probability that an alleged parent will be falsely included as a parent is estimated to be in the range of 1/716 to 1/2845.
Abstract: Six multiplexes developed for semiautomated fluorescence genotyping were evaluated for parentage testing. These multiplexes contained primer pairs for the amplification of 22 microsatellites on 17 bovine autosomes. Exclusion probabilities were determined from genotypes of 1022 Holstein cattle and 311 beef cattle belonging to five breeds. Two cases were considered: case 1, genotypes are known for an alleged parent and an offspring but genotypes of a confirmed parent are unknown; and case 2, genotypes are known for an alleged parent, a confirmed parent and an offspring. If the alleged parent is not the true parent, then the 22 markers will exclude the alleged parent with a probability of > 0.9986 for case 1 and with a probability of > 0.99999 for case 2. On the basis of these exclusion probabilities, the probability that an alleged parent will be falsely included as a parent is in the range of 1/716 to 1/2845 for case 1 and 1/1.2 million to 1/159753 for case 2. In addition to these results, a rapid and efficient non-organic method for extraction of DNA from semen is described.
123 citations
TL;DR: In this paper, two polyethylene glycols (PEG, M = 35,000) endcapped with short fluorocarbon tails were synthesized and characterized, and they associate strongly to form mic...
Abstract: Two polyethylene glycols (PEG, M = 35 000) end-capped with short fluorocarbon tails were synthesized and characterized. In aqueous solution, the fluorocarbon portions associate strongly to form mic...
116 citations
TL;DR: The anomalous migration of ssDNA fragments is used to optimize denaturation conditions for capillary electrophoresis and the sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C).
Abstract: Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.
95 citations
TL;DR: The isolation and cloning of a gRNA-interacting polypeptide from Trypanosoma brucei named gBP21 is described, which is arginine-rich and binds to gRNA molecules with a dissociation constant in the nanomolar range, and the gRNA/gBP21 ribonucleoprotein complex is more stable than naked guide RNAs.
TL;DR: In this article, a micromachined diffraction gratings were mounted on CCD imaging devices for high-dispersion and -sensitivity applications, and the other for low-cost consumer applications.
Abstract: Miniature spectrometers have been demonstrated by mounting micromachined diffraction gratings onto CCD imaging devices. Two implementations are tested: one for high-dispersion and -sensitivity applications, and the other for low-cost consumer applications. The first system shows a dispersion of 1.7 nm/pixel and a resolution of 74.4 for the bandwidth of interest. The free spectral range of the device is designed to be in the visible range for this particular application. The diffraction efficiency of the system is 63%. The second, low-cost system demonstrates a dispersion and resolution of 2.55 nm/pixel and 69.8, respectively. These specifications are comparable to that of a conventional, low-end commercial spectrometer. Results are shown for their applications in biochemical analysis. Further optimization is sought by adding micromachined lenses and creating specialized, computer-generated gratings to compress and shape the spectral signal.
TL;DR: Results indicate the feasibility of using the ribozyme technology to determine the roles of individual γ subunits in receptor-G protein-effector pathways in vivo, and point to a specific role of the γ7 subunit in the regulation of adenylylcyclase activity in response to isoproterenol.
TL;DR: The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5 to identify new alleles directly and an identical new "HLA-A*0103" was identified in two Caucasian samples.
TL;DR: TAMRA fluorescent dye-labeled non-nucleosidic synthesis supports have been derivatized for automated synthesis of 3′ dye- labeled oligonucleotides for Taqman assay and FRET experiments.
TL;DR: An automated online solid-phase synthesis of Ac-cac ct T-Gly-OH1 using standard DNA and appropriately protected PNA building blocks is described, which forms stable duplexes with complementary DNA and RNA.
TL;DR: Results of this first in depth survey of ground water across the United States confirm the ubiquity of Legionella in aquatic environments, even ground waters.
TL;DR: Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase by low concentrations of guanidine hydrochloride, and it is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.
Abstract: Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.
TL;DR: The rhesus ADR beta3-adrenergic receptor is more similar to the human ADRbeta3 than to the rodent ADRBeta3 suggesting that this primate model may be more appropriate for physiologic and therapeutic studies of the ADR Beta3 axis.
TL;DR: Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories, which is expected given the technical differences in the methodologies.
Abstract: The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories.
TL;DR: GPC3, the gene modified in the Simpson-Golabi-Behmel gigantism/overgrowth syndrome (SGBS), is shown to span more than 500 kb of genomic sequence, with the transcript beginning 197 bp 5' of the translational start site.
TL;DR: The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype, illustrating the complexity of this family.
TL;DR: It is suggested that PDIp, too, can be involved in completion of cotranslational as well as posttranslational translocation of proteins into mammalian microsomes.
01 Jan 1997
TL;DR: Modifications have included non-nucleosidic dye linkers, 2'-O-Me RNA substitutions, and pyrimidine C5-propyne substitutions.
Abstract: We have prepared and evaluated a series of structural analogues of TaqMan PCR probes in an effort to identify second-generation probes with improved physical properties and performance. Modifications have included non-nucleosidic dye linkers, 2'-O-Me RNA substitutions, and pyrimidine C5-propyne substitutions.
Journal Article•
TL;DR: This work has developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF, and combined well established PCR technology with Oligonucleotide Ligation Assay and Sequence-Coded Separation, two relatively new technologies.
Abstract: The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucl...
31 Mar 1997
TL;DR: In this paper, the authors mounted micromachined diffraction gratings onto CCD imaging devices for high dispersion and high sensitivity applications, and the other for low-cost consumer applications.
Abstract: Miniature spectrometers were demonstrated by mounting micromachined diffraction gratings onto CCD imaging devices. Two implementations were tested: one for high dispersion and high sensitivity applications, and the other for low-cost consumer applications. The first system showed a dispersion of 1.7 nm/pixel and a resolution of 74.4 for the bandwidth of interest. The free spectral range of these devices was designed to be in the visible range for this particular application. The diffraction efficiency of the system was 63%. The second, low-cost system demonstrated a dispersion and resolution of 2.55 nm/pixel and 69.8 respectively. These specifications are comparable to that of a conventional, low-end commercial spectrometer. Results are shown for their applications in biochemical analysis. Optimization was sought by adding micromachined lenses and creating specialized, computer generated gratings to compress and shape the spectral signal.
TL;DR: Fluorescent DDRT-PCR increases throughput and obviates the handling of hazardous radioisotopes, and a PCR cycling profile, expected to give improved reproducibility, is described.
Abstract: Differential display reverse transcription PCR (DDRT-PCR) is a procedure used to identify the induction or repression of gene e x pression. In most DDRT-PCR protocols, radioisotopes are incorpo rated during PCR and the cDNA products are detected by autoradio graphy. This report describes the fluorescent labeling of cDNAs and their detection on automated sequencers from PE Applied Biosy s tems. A fluorescent tag can be incorporated into the PCR product b y using either a labeled primer or a labeled dUTP. The fluorescent sig nals are analyzed by GENESCAN ™ software. Fluorescent DDRT PCR increases throughput and obviates the handling of hazardou s radioisotopes. A PCR cycling profile, expected to give improved r e producibility, is also described. Because amplified cDNAs can’t b e recovered from the automated sequencer gel, suggestions are given for the identification and recovery of differentially expressed cDNAs .
TL;DR: The artificial transposon, AT-2, a Bluescript derivative containing the dhfr gene and unique primer sites at both ends of the insertion DNA were utilized, which greatly simplified sequencing of regions that had been difficult to accomplish otherwise.
TL;DR: In this article, an automated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.
Abstract: Automated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.