The Experts below are selected from a list of 15375 Experts worldwide ranked by ideXlab platform
Paul D. Siebert - One of the best experts on this subject based on the ideXlab platform.
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Suppression Subtractive Hybridization.
Methods of Molecular Biology, 2004Co-Authors: Denis V. Rebrikov, Sejal Desai, Paul D. Siebert, Sergey LukyanovAbstract:Suppression Subtractive Hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. To our knowledge, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. It is based primarily on a suppression polymerase chain reaction (PCR) technique (described narrowly in Chapter 3) and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs or genomic DNA fragments and simplifies analysis of the subtracted library. SSH technique is applicable to many comparative and functional genetic studies for the identification of disease, developmental, tissuespecific, or other differentially expressed genes, as well as for the recovery of genomic DNA fragments distinguishing the samples under comparison. This chapter provides an insight into SSH practical use and contains detailed protocol for generation of subtracted cDNAs (which is the most frequent SSH application) and differential screening of the resulting subtracted cDNA library. As shown in many examples, the SSH technique may result in over 1000-fold enrichment for rare sequences in a single round of Subtractive Hybridization. Finally, we discuss the characteristics of cDNA-subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as procedure for rapid identification of truly differentially expressed cDNA clones.
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20 suppression Subtractive Hybridization a versatile method for identifying differentially expressed genes
Methods in Enzymology, 1999Co-Authors: Luda Diatchenko, Sergey Lukyanov, Paul D. SiebertAbstract:Abstract A new and highly effective method, termed suppression Subtractive Hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of Subtractive Hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a Hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.
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pcr based Subtractive Hybridization and differences in gene content among strains of helicobacter pylori
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Natalia S Akopyants, Paul D. Siebert, Sergey Lukyanov, Luda Diatchenko, Arkady F Fradkov, Jason Hill, E D Sverdlov, Douglas E BergAbstract:Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based Subtractive Hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by Subtractive Hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot Hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.
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suppression Subtractive Hybridization a method for generating differentially regulated or tissue specific cdna probes and libraries
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Luda Diatchenko, Sergey Lukyanov, E D Sverdlov, Aaron P Campbell, Alex Chenchik, Fauzia Moqadam, Betty C B Huang, Konstantin A Lukyanov, Nadya G Gurskaya, Paul D. SiebertAbstract:Abstract A new and highly effective method, termed suppression Subtractive Hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of Subtractive Hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a Hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.
Sergey Lukyanov - One of the best experts on this subject based on the ideXlab platform.
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Suppression Subtractive Hybridization.
Methods of Molecular Biology, 2004Co-Authors: Denis V. Rebrikov, Sejal Desai, Paul D. Siebert, Sergey LukyanovAbstract:Suppression Subtractive Hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. To our knowledge, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. It is based primarily on a suppression polymerase chain reaction (PCR) technique (described narrowly in Chapter 3) and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs or genomic DNA fragments and simplifies analysis of the subtracted library. SSH technique is applicable to many comparative and functional genetic studies for the identification of disease, developmental, tissuespecific, or other differentially expressed genes, as well as for the recovery of genomic DNA fragments distinguishing the samples under comparison. This chapter provides an insight into SSH practical use and contains detailed protocol for generation of subtracted cDNAs (which is the most frequent SSH application) and differential screening of the resulting subtracted cDNA library. As shown in many examples, the SSH technique may result in over 1000-fold enrichment for rare sequences in a single round of Subtractive Hybridization. Finally, we discuss the characteristics of cDNA-subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as procedure for rapid identification of truly differentially expressed cDNA clones.
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a new planarian extrachromosomal virus like element revealed by Subtractive Hybridization
Molecular Biology, 2002Co-Authors: D V Rebrikov, E A Bogdanova, Maria E Bulina, Sergey LukyanovAbstract:A combination of suppression Subtractive Hybridization and a new technique of mirror orientation selection was used to compare the total DNA for two, sexual and asexual, races of freshwater planarian Girardia tigrina. Several race-specific DNA fragments were found. A new element termed planarian extrachromosomal virus-like element (PEVE) was revealed in the asexual race. The PEVE genome contains two unique regions, Ul and Us, which are flanked by inverted repeats. Two variants observed for the PEVE genome differ in combination of single- and double-stranded regions corresponding to Ul and Us. The PEVE genome codes for two helicases, one homologous to the circovirus replication initiation protein (Rep) and one corresponding to the helicase domain of papillomavirus E1. PEVE is nonuniformly distributed through the planarian body and is possibly replicated only in certain parenchymal cells.
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20 suppression Subtractive Hybridization a versatile method for identifying differentially expressed genes
Methods in Enzymology, 1999Co-Authors: Luda Diatchenko, Sergey Lukyanov, Paul D. SiebertAbstract:Abstract A new and highly effective method, termed suppression Subtractive Hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of Subtractive Hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a Hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.
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pcr based Subtractive Hybridization and differences in gene content among strains of helicobacter pylori
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Natalia S Akopyants, Paul D. Siebert, Sergey Lukyanov, Luda Diatchenko, Arkady F Fradkov, Jason Hill, E D Sverdlov, Douglas E BergAbstract:Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based Subtractive Hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by Subtractive Hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot Hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.
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ISMB - The Mathematical Model of Subtractive Hybridization and Its Practical Application
Proceedings. International Conference on Intelligent Systems for Molecular Biology, 1996Co-Authors: Olga D. Ermolaeva, Sergey Lukyanov, Eugene D. SverdlovAbstract:A novel theory of Subtractive Hybridization including (or based on) the kinetic model of this process was proposed. A computer program modeling the process of subtraction was developed. Basing on the theory, a novel method of Subtractive Hybridization was proposed allowing routine comparison of genomes and products of genome expression. The method was applied to studies of the genetic mechanisms of embryogenesis, regeneration, cell differentiation and tumor transformation.
Luda Diatchenko - One of the best experts on this subject based on the ideXlab platform.
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20 suppression Subtractive Hybridization a versatile method for identifying differentially expressed genes
Methods in Enzymology, 1999Co-Authors: Luda Diatchenko, Sergey Lukyanov, Paul D. SiebertAbstract:Abstract A new and highly effective method, termed suppression Subtractive Hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of Subtractive Hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a Hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.
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pcr based Subtractive Hybridization and differences in gene content among strains of helicobacter pylori
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Natalia S Akopyants, Paul D. Siebert, Sergey Lukyanov, Luda Diatchenko, Arkady F Fradkov, Jason Hill, E D Sverdlov, Douglas E BergAbstract:Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based Subtractive Hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by Subtractive Hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot Hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.
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suppression Subtractive Hybridization a method for generating differentially regulated or tissue specific cdna probes and libraries
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Luda Diatchenko, Sergey Lukyanov, E D Sverdlov, Aaron P Campbell, Alex Chenchik, Fauzia Moqadam, Betty C B Huang, Konstantin A Lukyanov, Nadya G Gurskaya, Paul D. SiebertAbstract:Abstract A new and highly effective method, termed suppression Subtractive Hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of Subtractive Hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a Hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.
David Murphy - One of the best experts on this subject based on the ideXlab platform.
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Suppression Subtractive Hybridization.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Mohamed T Ghorbel, David MurphyAbstract:Comparing two RNA populations that differ from the effects of a single independent variable, such as a drug treatment or a specific genetic defect, can establish differences in the abundance of specific transcripts that vary in a population dependent manner. There are different methods for identifying differentially expressed genes. These methods include microarray, Serial Analysis of Gene Expression (SAGE), and quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Herein, the protocol describes an easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under examination. It is specifically relevant when low levels of RNA starting material are available. This protocol describes the use of Switching Mechanism At RNA Termini Polymerase Chain Reaction (SMART-PCR) to amplify cDNA from small amounts of RNA. The amplified cDNA populations under comparison are then subjected to Suppression Subtractive Hybridization (SSH-PCR). SSH-PCR is a technique that couples Subtractive Hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The resulting products are cDNA populations enriched for significantly overrepresented transcripts in either of the two input RNAs. These cDNA populations can then be cloned to generate subtracted cDNA library. Microarrays made with clones from the subtracted forward and reverse cDNA libraries are then screened for differentially expressed genes using targets generated from tester and driver total RNAs.
Patric A. Clapshaw - One of the best experts on this subject based on the ideXlab platform.
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Spinal Cord Transcriptome Analysis Using Suppression Subtractive Hybridization and Mirror Orientation Selection
Cellular and Molecular Neurobiology, 2006Co-Authors: Kanan B. Lathia, Patric A. ClapshawAbstract:Comparison of cDNA libraries derived from the spinal cord with those derived from the visual cortex by means of forward and reverse Subtractive Hybridization resulted in the cataloguing of 60 genes differentially expressed in the spinal cord. 1. The differentially expressed genes represent a mixture of novel and known sequences with known and unknown protein products. 2. The possibility that the subtraction process was simply overwhelmed by background sequences was significantly reduced by several observations including comparisons between suppression Subtractive Hybridization (SSH) and mirror orientation selection (MOS). 3. Nearly half of all genes up-regulated in the spinal cord are of myelin origin. 4. Twenty-five percent of all up-regulated clones in the spinal cord versus the visual cortex are for proteolipid protein. 5. Ten percent of all up-regulated clones in spinal cord versus visual cortex are for ferretin heavy chain, which is known to be produced in oligodendroglial cells in the CNS. 6. Two of the up-regulated sequences, proteolipid protein and N-myc down-regulated gene 4, are identified with genes known to directly affect neuron survival. 7. Two of the up-regulated genes, ferritin and transferrin, are indirectly associated with apoptosis through their ability to sequester iron and reduce free radical formation.
