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丁香实验推荐阅读
Computer Analysis of Cloned Sequences

Analysis of the data generated from cDNA sequences, an important step in the final stages of any sequencing project, can provide insights into gene structure and function as well as help to direct the experimental approaches to obtaining the sequence of a full-length clone even when only prelim ...

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Isolation of Genetic Suppressor Elements (GSEs) from Random Fragment cDNA Libraries in Retroviral Vectors

Cellular phenotypes resulting from decreased function of a specific gene are manifested as recessive, since they are suppressed in the presence of a normal allele of the corresponding gene. The powerful gene-transfer techniques, which have played a key role in the studies of dominant pheno ...

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Expression and Preparation of Fusion Proteins from Recombinant gt1.1 Phages

The phage λgt1.1 system has become increasingly popular for expression of cDNAs or genomic DNAs either in phage plaques or in bacteria lysogenized with recombinant phages (1,2). It offers the advantages of high cloning efficiency, high-level expression, the relative stability of β-gala ...

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Protein Interaction Cloning by Far-Western Screening of -Phage cDNA Expression Libraries

One of the most powerful ways to gain novel insights into a particular protein’s cellular functions is to identify other proteins with which it specifically interacts. Traditionally, this problem has been approached by use of protein biochemistry, involving purification of interact ...

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Evolution of MicroRNAs

MicroRNAs (miRNAs) form a large class of small regulatory RNAs in eukaryotes. Although they share a common processing pathway and certain structural features, in general, there is no detectable sequence similarity among miRNAs from a given organism. On the other hand, many miRNAs are members ...

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Structure Analysis of MicroRNA Precursors

MicroRNA biogenesis occurs in several steps from their precursors having irregular hairpin structures. The highly variable architecture of these stem-and-loop structures, which have terminal loops of various sizes and diverse structure destabilizing motifs present in their ...

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Perspectives

The discoveries of microRNAs (miRNAs), a class of noncoding RNAs that can regulate gene expression by translational repression, have opened a new avenue on gene modulation in mammalian cells. Conceivably, this highly effective method of modulation of gene expression would be exploited ...

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MicroRNA Biogenesis: Isolation and Characterization of the Microprocessor Complex

The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that have been implicated in controlling diverse pathways in a variety of organisms (1,2). For posttranscriptional gene silencing, one strand of the miRNA is used to guide components of the RNA interfere ...

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Recognition and Cleavage of Primary MicroRNA Transcripts

MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary miRNAs (pri- ...

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MicroRNAs and Messenger RNA Turnover

RNA-induced silencing complexes (RISCs) are multiple-turnover entities that direct many rounds of site-specific target mRNA cleavage (1). A principal RISC component in all eukaryotes is a member of the Argonaute (AGO) protein family (4). AGO contains the conserved PAZ and PIWI domains, and ...

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Mouse Embryonic Stem Cells as a Model Genetic System to Dissect and Exploit the RNA Interference Machinery

Conditional gene targeting is often a useful approach to elucidate the in vivo function of a gene. We use this approach to investigate the biological role of the RNA interference (RNAi) pathway in mammals. In addition, the RNAi machinery in mammalian cells can be exploited for gene knock-down expe ...

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Prediction of MicroRNA Targets

I describe the use of RNAhybrid, a program that predicts multiple potential binding sites of microRNAs (miRNAs) in large target RNAs. The core algorithm finds the energetically most favorable hybridization sites of a miRNA in a large potential target RNA. Intramolecular hybridization ...

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Complications in Mammalian MicroRNA Target Prediction

In this chapter, we review evidence that at least three different types of microRNA (miRNA)-messenger RNA (mRNA) target interactions exist in mammals: short seeds, long seeds, and “perfect” hits (allowing G:U matches). Because new types of miRNAs are still being discovered, this list may not yet be ...

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Prediction of Human MicroRNA Targets

MicroRNAs (miRNAs) are small, nonprotein-coding RNAs that regulate gene expression. Although hundreds of human miRNA genes have been discovered, the functions of most of these are unknown. Computational predictions indicate that miRNAs, which account for at least 1% of human protein- ...

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The Tetramethylammonium Chloride (TMAC) Method for Screening cDNA Libraries with Highly Degenerate Oligonucleotide Probes Obtained by Reverse Translat

If an unknown protem is purified and available in relatively small amounts, it is possible to determine the sequences of short internal peptides (1). In order to determine the whole sequence of the protein by cDNA cloning, one of the peptides of perhaps five to seven amino acids may be reverse translated ...

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Preparation of Competent Cells for High-Efficiency Plasmid Transformation of Escherichia coli

Transformation of Escherichia coli was first described by Mandel and Higa (1), who reported that E. coli cells, after treatment with calcium chloride, can take up bacteriophage λ DNA and produce viable phage particles. The conditions for the transfer of exogenous DNA into E. coli have been examin ...

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Cloning Sequence-Specific DNA-Binding Factors from cDNA Expression Libraries Using Oligonucleotide Binding Site Probes

The method described in this chapter has been used in the molecular cloning of transcription factors and other factors with DNA-binding activity toward specific double-stranded DNA sequences. The protocol is based on the method of Singh et al. (1) and shares feature with the immunological ap ...

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Cycle Sequencing of Polymerase Chain Reaction-Amplified Genomic DNA with Dye-Labeled Universal Primers

Cycle sequencing of polymerase chain reaction (PCR) products has become the method of choice for sequencing known genomic DNA fragments. When fluorescent DNA sequencers are used, this requires the incorporation of a fluorescent dye into the cycle sequencing product either by using lab ...

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Analysis of Nucleic Acids by Tandem Hybridization on Oligonucleotide Microarrays

Oligonucleotide arrays (also known as DNA chips, gene chips, or genosensor chips) are emerging as a powerful research tool in nucleic acid sequence analysis. Several technical challenges remain to be solved, however, before oligonucleotide arrays can reach their full potential and be im ...

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DNA Sequencing by Hybridization with Arrays of Samples or Probes

This chapter focuses on sequencing by hybridization (SBH), an advanced DNA sequencing technique first proposed in 1987 (1). SBH procedures determine DNA sequence information by screening DNA oligomers (typically 7-to 11-mers) for their ability to hybridize with target DNA. The set of ove ...

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