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siRNA Delivery In Vivo

Inhibition of gene expression at the mRNA levels can be accomplished by several methods, including ribozymes, DNAzymes, and small interfering RNAs (siRNAs) (1–3). This is now driven predominantly by siRNAs, as they are not technically demanding as traditional antisense and ribozyme tec ...

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Immunoprecipitation of MicroRNPs and Directional Cloning of MicroRNAs

MicroRNAs (miRNAs) comprise a class of approx 22-nucleotide (nt) regulatory RNAs, found in plants and animals (1–8). miRNAs contain 5′ phosphates and 3′ hydroxyls and are processed by the Dicer nuclease from one of the stems of longer precursors (pre-miRNAs) that form stem-loop structures (9–11 ...

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Exon-Specific RNA Interference: A Tool to Determine the Functional Relevance of Proteins Encoded by Alternatively Spliced mRNAs

The majority of metazoan genes encode pre-mRNAs that are subject to alternative splicing. For example, it has recently been estimated that as many as 74% of human genes encode alternatively spliced mRNAs (1). An alternatively spliced gene can generate anywhere from 2 different isoforms to as ma ...

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Detection of MicroRNAs and Assays to Monitor MicroRNA Activities In Vivo and In Vitro

MicroRNAs (miRNAs) are approx 22-nucleotide (nt) regulatory RNAs derived from endogenous genes and processed from longer (approx 70 nt, in animals) precursor RNAs (pre-miRNAs) (1–8). miRNAs bind to Argonaute (Ago) proteins, such as Ago-2 (also known as eIF2C2) (6,9), and typically associate ...

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High-Throughput Analysis of MicroRNA Gene Expression Using Sensitive Probes

MicroRNAs (miRNAs) represent a new class of noncoding RNAs whose functions are, in most cases, unknown, but are believed to play important biological roles (1). These tiny RNAs are genome encoded as primary transcripts, referred to as pri-miRNAs, which are processed in the nucleus by Dorsha, a ribo ...

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Identification of Components of RNAi Pathways Using the Tandem Affinity Purification Method

RNA interference (RNAi) is rapidly becoming a standard laboratory technique for understanding and regulating the function of specific genes in evolutionarily diverse organisms, including plants, Caenorhabditis elegans, Drosophila, and mammalian cells (1–10). RNAi is initi ...

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Modification of Human U1 snRNA for Inhibition of Gene Expression at the Level of Pre-mRNA

U1 snRNA is a component of the U1 snRNP complex, which contains seven common snRNP proteins and three specific U1 snRNP proteins (1). It initiates spliceosome association with pre-mRNA by defining the 3′ boundary of exons (2). As the splicing reaction proceeds, U1 snRNP and the other spliceosome com ...

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Separation of Drosophila RNA Silencing Complexes by Native Gel Electrophoresis

Large, multicomponent complexes mediate many stages of eukaryotic gene expression, from transcription to translation. Despite their size, these complexes and their precursors can often be resolved and analyzed by native gel electrophoresis. Although other techniques exist f ...

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31 Inverse PCR: Genomic DNA Cloning

Inverse PCR (IPCR) was designed for amplifying anonymous flanking genomic DNA regions (1 2). The technique involves the digestion of source DNA, circulation of restriction fragments, and amplification using oligonucleotides that prime the DNA synthesis directed away from the core r ...

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Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers

Isolation of a full-length gene and analysis of expression profiling are fundamental and challenging in the current molecular biology. A great deal of effort is needed to detect unknown gene sequences by screening cDNA or genomic libraries by nucleic acid or protein probes. As the complete ge ...

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The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR

Because of its versatility, the Tn5 transposon has become a powerful tool in the classical genetic studies of Gram-negative bacteria. The Tn5 transposon is functional in a broad range of Gram-negative bacteria and transposes at a high frequency with low specificity of insertion (1,2), allow ...

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Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis

Current and recent efforts to determine the genomic DNA sequence for numerous organisms (e.g., Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Arabidopsis thaliana, Zea mays, Caenorhabditis elegans, Mus musculus, Homo sapiens, Schizosaccharomyces pombe, D ...

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Isolation of Large-Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR

Large insert libraries are critical in genome-related research. Bacterial artificial chromosome (BAC) libraries are widely used in plant, animal, and human research; the ends of BAC clones are used as probes for chromosome walking and to confirm overlapping of contigs, as well as RFLP marke ...

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Use of PCR in Library Screening: An Overview

Traditionally, libraries are screened with different probes to isolate target genes or sequences. These probes can be a particular sequence such as a cDNA, a polymerase chain reaction (PCR) product, or a genomic fragment (1). An oligonucleotide can be a probe if no closely related cDNAs or gene clo ...

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A “Step Down” PCR-Based Technique for Walking Into and the Subsequent Direct-Sequence Analysis of Flanking Genomic DNA

The hunt for missing sequence data, whether it be in pursuit of full-length clones or for promoter sequences, can be laborious and expensive. Indeed, protracted efforts to find missing sequences by library screening can be fraught with the frustration of foraging through libraries that may l ...

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Cloning of Homologous Genes by Gene-Capture PCR

Conventional procedures to isolate a gene belonging to an ortholog family usually imply the use or the construction of double-stranded cDNA libraries derived from a specific mRNA source of interest (cells or tissues) (1). The double-stranded DNA library plated on various membranes is scr ...

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Rapid and Nonradioactive Screening of Recombinant Libraries by PCR

The advent of the polymerase chain reaction (PCR) has greatly facilitated the isolation and characterization of clones from both cDNA and genomic libraries (1-3). Given the complexity of the genome of a particular organism or the relative abundance of a particular mRNA, within the cell type fr ...

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Rapid cDNA Cloning by PCR Screening (RC-PCR)

Now that the draft sequence of the human genome is available (1,2), cDNA cloning based on its sequence from a library is no longer an experimental goal, but a starting point and a routine laboratory practice. Hybridization screening with either radio-labeled or nonradio-labeled probes had been ...

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Generation and PCR Screening of Bacteriophage Sublibraries Enriched for Rare Clones (the Sublibrary Method )

The simple sublibrary method described in this chapter allows the detection and rapid isolation of rare clones from bacteriophage λ libraries. The method is based on the ability of polymerase chain reaction (PCR) to detect clones present in a library at very low frequencies. Clones present at f ...

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PCR-Based Screening for Bacterial Artificial Chromosome Libraries

The bacterial artificial chromosome (BAC) (1) is now the most widely utilized vector system to construct large-insert genomic libraries for genome analysis. It has the following advantages over another vector system, the yeast artificial chromosome (YAC) (2): ease of DNA purification, a ...

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