Showing papers by "Michael McClelland published in 1999"

Differential Display Probes for cDNA Arrays

Abstract: PCR with a combination of one arbitrar y and one oligo(dT) anchor primer can b e used to generate an effective probe fo r cDNA arrays. The method uses less than 1/200 of the amount of RNA used in som e other array hybridization methods. Each fin gerprint detects approximately 5% of th e transcribed mRNAs, sampled almost ind e pendent of abundance, using inexpensive E . col icolony arrays of expressed sequence tag (EST) clones. It proved necessary to alte r the differential display (DD) protocol to generate a sufficient mass of PCR product s for use as a probe. The use of different ol i go(dT) anchor primers with the same arb i trary primer resulted in considerable ove r lap among the genes sampled by each probe . This can be avoided by using different arb i trary primers with each oligo(dT) ancho r primer. Four genes not previously known to be regulated by epidermal growth facto r (EGF) and three genes known to be regula t ed by EGF in other cell types were chara c terized using DD fingerprints as probes fo r arrays. It should be possible to conver t archived DD fingerprints into effectiv e probes for arrays, allowing thousands of e x periments that have already been performed to yield further information. The use of DD fingerprints as probes should increase th e rate of identification of differentially regula t ed genes several fold while obviating th e need for cloning and sequencing .

Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts

Abstract: Our aim was to identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with rheumatoid arthritis (RA). In RA, amplification of a distinct PCR product suitable for sequencing could be observed. Sequence analysis identified the PCR product as highly homologous to a 434 base pair segment of the human centromere kinesin-like protein CENP-E. Differential expression of CENP-E was confirmed by quantitative reverse transcription PCR, immunohistochemistry and in situ hybridization. CENP-E expression was independent from prednisolone and could not be completely inhibited by serum starvation. RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. Also, because motifs of CENP-E show homologies to jun and fos oncogene products and are involved in virus assembly, CENP-E may be involved in the pathophysiology of RA.

Reduced complexity nucleic acid targets and methods of using same

Abstract: The invention provides a method of measuring the level of two or more nucleic acid molecules in a target by contacting a probe with a target comprising two or more nucleic acid molecules, wherein the nucleic acid molecules are arbitrarily sampled and wherein the arbitrarily sampled nucleic acid molecules comprise a subset of the nucleic acid molecules in a population of nucleic acid molecules; and detecting the amount of specific binding of the target to the probe. The invention also provides a method of measuring the level of two or more nucleic acid molecules in a target by contacting a probe with a target comprising two or more nucleic acid molecules, wherein the nucleic acid molecules are statistically sampled and wherein the statistically sampled nucleic acid molecules comprise a subset of the nucleic acid molecules in a population of nucleic acid molecules; and detecting the amount of specific binding of the target to the probe.

Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome

Abstract: As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome. The average insert size is 17 kb. Sequences were obtained from both ends of each clone in this set. A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank. This resource of clones is available from the Salmonella Genome Stock Center. A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes. Fully 30% of the S. typhimurium sequences have no close homologs in the GenBank database.

Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction.

Abstract: Publisher Summary Genomic fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.

Reduced complexity probes for DNA arrays.

Abstract: Publisher Summary There have been many advances in methods to manufacture and probe arrays of DNAs. Membranes with hundreds or thousands of polymerase chain reaction (PCR) products from cDNA clones are commercially available. Any cDNA clones on the array that are from the most abundant few thousand of mRNAs in a cell can easily be detected using a radiolabeled probe containing the full complexity of the mRNA population in the cell. The only limitation is background hybridization to all clones. This chapter explains a strategy based on cDNA fingerprints generated by RNA arbitrarily primed PCR (RAP-PCR). This method generates different subsets of the mRNA population, depending on the primers used. Some of the mRNAs that are difficult to detect using total cDNA probes are sufficiently represented in these reduced complexity probes to be easily detected on arrays of colonies. This method has the further advantage that it generates a labeled probe using hundreds-fold less RNA than is currently used by any other array probing methods. The chapter discusses the use of statistically primed PCR (SP-PCR) to generate probes.

Analyse des Genexpressionsmusters von synovialen Fibroblasten von Patienten mit rheumatoider Arthritis mittels RAP-PCR Differential Display

Abstract: □ Fragestellung Im rheumatischen Gelenk finden sich transformiert erscheinende Fibroblasten an der Invasionszone in den Knorpel und Knochen. Uber die Faktoren, die zu diesem aggressiven Phanotyp fuhren, ist bisher wenig bekannt. Ziel der Untersuchungen war daher, mittels RAP-PCR (RNA arbitrarily primed PCR) zwischen rheumatoiden Fibroblasten und Osteoarthritis-Fibroblasten differentiell exprimierte Gene zu erfassen.

Screening differentially displayed PCR products by single-strand conformation polymorphism gels

Abstract: Publisher Summary Differential display and ribonucleic acid (RNA) arbitrarily primed PCR have been extensively used to investigate differential gene expression and coordinate regulation in a wide variety of situations, including cell responses to different treatments and growth conditions and comparisons of different developmental stages. The RNA fingerprinting approach has also found many applications in cancer research. Several modifications to the original protocols have been reported, and the development of new technologies, such as automated sequencing or the use of fluorescent-tagged primers, is facilitating the use of this approach RNA fingerprinting has three steps: (i) cDNA synthesis, (ii) isolation and characterization of differentially amplified transcripts, and (iii) confirmation of differential expression. The step of isolation and characterization of the PCR fragments representing differentially amplified products is often a problem. This is so because it's laborious or gives products that are not differentially expressed because they are not the ones being targeted. These problems are associated with the comigration of other PCR products in the fingerprint gel. This chapter focuses on the use of single-strand polymorphism gels to facilitate the isolation and purification of differentially amplified cDNAs from differential display and RAP-PCR fingerprinting experiments. The two protocols that are currently in use are also provided.

Cibles d'acide nucleique de moindre complexite et leurs methodes d'utilisation

Abstract: La presente invention concerne une methode pour mesurer le niveau de deux molecules d'acide nucleique ou plus dans une cible par mise en contact d'une sonde et d'une cible renfermant au moins deux molecules d'acide nucleique. Ces molecules d'acide nucleique sont prelevees de facon arbitraire et renferment un sous-ensemble de molecules d'acide nucleique dans une population de molecules d'acide nucleique. Cette methode permet egalement de detecter l'importance de la liaison specifique entre la cible et la sonde. De plus, l'invention concerne une methode pour mesurer le niveau de deux molecules d'acide nucleique ou plus dans une cible par mise en contact d'une sonde et d'une cible renfermant au moins deux molecules d'acide nucleique. Ces molecules d'acide nucleique sont prelevees de facon statistique et renferment un sous-ensemble de molecules d'acide nucleique dans une population de molecules d'acide nucleique. Cette methode permet egalement de detecter l'importance de la liaison specifique entre la cible et la sonde.