Preparation and Use of Nonradioactive Hybridization Probes

1998 ◽  
pp. 377-384 ◽  
Author(s):  
Victor T.-W. Chan
Keyword(s):  
Hybridization Probes

scholarly journals Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 681-698 ◽  
Author(s):  
A J Link ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Physical Map ◽  
Single Copy ◽  
Restriction Endonucleases ◽  
Yeast Genome ◽  
Hybridization Probes ◽  
Copy Dna ◽  
Single Copy Dna

Abstract A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA

1987 ◽  
Vol 142 (2) ◽  
pp. 519-526 ◽  
Author(s):  
Avi Reisfeld ◽  
Jeffrey M. Rothenberg ◽  
Edward A. Bayer ◽  
Meir Wilchek
Keyword(s):  
Hybridization Probes

Specific hybridization probes for mouse α2(IX) and α1(X) collagen mRNAs

1992 ◽  
Vol 1130 (1) ◽  
pp. 78-80 ◽  
Author(s):  
Kati Elima ◽  
Marjo Metsäranta ◽  
Johanna Kallio ◽  
Merja Perälä ◽  
Iiro Eerola ◽  
...  
Keyword(s):  
Hybridization Probes ◽  
Specific Hybridization

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 317-326
Author(s):  
M. Caizergues-Ferrer ◽  
C. Mathieu ◽  
P. Mariottini ◽  
F. Amalric ◽  
F. Amaldi
Keyword(s):  
Ribosomal Rna ◽  
Rna Processing ◽  
Protein Detection ◽  
Developmental Expression ◽  
Oocyte Growth ◽  
Small Nuclear Ribonucleoprotein ◽  
Hybridization Probes ◽  
Ribosomal Rna Processing ◽  
Protein Components ◽  
Nuclear Ribonucleoprotein

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.

Escherichia coli phenylalanyl-tRNA synthetase operon: transcription studies of wild-type and mutated operons on multicopy plasmids

1982 ◽  
Vol 152 (2) ◽  
pp. 661-668
Author(s):  
J A Plumbridge ◽  
M Springer
Keyword(s):  
Structural Gene ◽  
Trna Synthetase ◽  
Mrna Levels ◽  
Structural Genes ◽  
Cis Acting ◽  
Trna Synthetases ◽  
Hybridization Probes ◽  
Steady State Levels ◽  
Derivatives Of

The construction of three lambda bacteriophages containing parts of the structural gene for threonyl-tRNA synthetase, thrS, and those for the two subunits of phenylalanyl-tRNA synthetases, pheS and pheT, is described. These phages were used as hybridization probes to measure the in vivo levels of mRNA specific to these three genes. Plasmid pB1 carries the three genes thrS, pheS, and pheT, and strains carrying the plasmid show enhanced levels of mRNA corresponding to these genes. Although the steady-state levels of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase produced by the presence of the plasmid differed by a factor of 10, their pulse-labeled mRNA levels were about the same. Mutant derivatives of pB1 were also analyzed. Firstly, a cis-acting insertion located before the structural genes for phenylalanyl-tRNA synthetase caused a major decrease in both pheS and pheT mRNA. Secondly, mutations affecting either structural gene pheS or pheT caused a reduction in the mRNA levels for both pheS and pheT. This observation suggests that autoregulation plays a role in the expression of phenylalanyl-tRNA synthetase.

Human actin genes are single copy for alpha-skeletal and alpha-cardiac actin but multicopy for beta- and gamma-cytoskeletal genes: 3' untranslated regions are isotype specific but are conserved in evolution

1983 ◽  
Vol 3 (10) ◽  
pp. 1783-1791
Author(s):  
P Ponte ◽  
P Gunning ◽  
H Blau ◽  
L Kedes
Keyword(s):  
Human Genome ◽  
Single Copy ◽  
Untranslated Regions ◽  
Muscle Actin ◽  
Actin Genes ◽  
Hybridization Probes ◽  
Single Exception ◽  
Multiple Copies ◽  
Human Genomic ◽  
Unambiguous Assignment

We have constructed isotype-specific subclones from the 3' untranslated regions of alpha-skeletal, alpha-cardiac, beta-cytoskeletal, and gamma-cytoskeletal actin cDNAs. These clones have been used as hybridization probes to assay the number and organization of these actin isotypes in the human genome. Hybridization of these probes to human genomic actin clones (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981; Engel et al., Mol. Cell. Biol. 2:674-684, 1982) has allowed the unambiguous assignment of the genomic clones to isotypically defined actin subfamilies. In addition, only one isotype-specific probe hybridizes to each actin-containing gene, with a single exception. This result suggests that the multiple actin genes in the human genome are not closely linked. Genomic DNA blots probed with these subclones under stringent conditions demonstrate that the alpha-skeletal and alpha-cardiac muscle actin genes are single copy, whereas the cytoskeletal actins, beta and gamma, are present in multiple copies in the human genome. Most of the actin genes of other mammals are cytoplasmic as well. These observations have important implications for the evolution of multigene families.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

Mitochondrial DNA polymorphism in a black fly, Simulium vittatum (Diptera: Simuliidae)

1998 ◽  
Vol 76 (3) ◽  
pp. 440-447 ◽  
Author(s):  
X Zhu ◽  
K P Pruess ◽  
T O Powers
Keyword(s):  
New York ◽  
Mitochondrial Dna ◽  
Rflp Analysis ◽  
Mitochondrial Haplotype ◽  
Black Flies ◽  
Mitochondrial Dna Polymorphism ◽  
Simulium Vittatum ◽  
Hybridization Probes ◽  
Restriction Sites ◽  
Mitochondrial Heterogeneity

Mitochondrial DNA (mtDNA) was extracted from pooled field-collected samples representing six species of black flies (Cnephia dacotensis, Simulium bivittaum, S. johansenni, S. luggeri, S. piperi, S. vittatum) and compared by restriction fragment length polymorphism (RFLP) analysis. Morphospecies were molecularly distinct, with few shared restriction fragments. Eleven populations of S. vittatum were found that appeared to be homogeneous for a single mitochondrial haplotype. Ten other populations of S. vittatum showed extensive mitochondrial heterogeneity. In part, these samples contained mixtures of two cytologically recognized siblings: IIIL-1 and IS-7. About 70% of the mitochondrial genome of a population pure for sibling IIIL-1 was cloned as five HindIII fragments, which were used as hybridization probes to examine individual black flies. Thirteen mtDNA haplotypes involving permutations of 10 HindIII restriction sites were identified in individual black flies examined from 26 populations. DNA from 168 larvae cut with both EcoR1 and HindIII revealed five additional haplotypes. One HindIII haplotype was present in 84% of 390 larvae examined and predominated in every population examined from New York to California and in both the IIIL-1 and IS-7 siblings. Nebraska populations had individuals with nearly all known haplotypes. The most common haplotype was usually the only form present in warm, silty streams with organic enrichment. Rarer haplotypes were found in cool, spring-fed streams but without clear geographic or phylogenetic components.

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