生物化学技术

Genome browsers are important tools for studying genomes given the vast amounts of data available. This chapter focuses on providing the reader with the skills necessary to perform relatively simple, yet powerful, analysis relating to the structure of the transcription unit. Studying a ...

This chapter describes a method for the isolation of intact polyadenylated mRNA using LNA oligo(T) capture. The method enables efficient isolation of poly(A)+ RNA directly from guanidinium thiocyanate (GuSCN)-containing cell or tissue extract by combining the design of biotinylat ...

In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in μg to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter seq ...

Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when possi ...

The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein codi ...

The traditional method for determining the transcription rate of a gene, nuclear run-on, is time consuming, laborious, and involves the use of high levels of radio-labeled nucleotides. When combined with measurements of mRNA levels, RNA polymerase II (Pol II) chromatin immunoprecipit ...

Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify ...

RNAi is now the preferred method for silencing gene expression in a variety of systems. In this chapter we describe the procedure for applying short-hairpin RNA (shRNA) to study gene function. Detailed descriptions of target site selection, shRNA construction, shRNA transfection and tar ...

Small interfering RNA (siRNAs), the main effector of RNA interference (RNAi), are now routinely used to assess gene function, both in vitro and in vivo, and many innovative screens have been reported on the use of RNAi to identify potential drug targets. Despite several technical advances, howev ...

The characterization of alternatively spliced RNA is a frequently performed task in the molecular biology laboratory. Several methods have been established to characterize specific transcripts, of which microarrays, northern analysis, RT-PCR and nuclease protection assa ...

A rapidly increasing number of human diseases are now recognized as being caused by the selection of wrong splice sites. In most cases, these changes in alternative splice site selection are due to single nucleotide exchanges in splicing regulatory elements. This chapter describes the use of ...

Pfizer Global Virtual Library (PGVL) of 1013 readily synthesizable molecules offers a tremendous opportunity for lead optimization and scaffold hopping in drug discovery projects. However, mining into a chemical space of this size presents a challenge for the concomitant design in ...

Fragment-based drug design (FBDD), which is comprised of both fragment screening and the use of fragment hits to design leads, began more than 15 years ago and has been steadily gaining in popularity and utility. Its origin lies on the fact that the coverage of chemical space and the binding efficiency ...

A successful fragment-based lead discovery (FBLD) campaign largely depends on the content of the fragment collection being screened. To design a successful fragment collection, several factors must be considered, including collection size, property filters, hit follow-up cons ...

Structure-based library design employs both structure-based drug design (SBDD) and combinatorial library design. Combinatorial library design concepts have evolved over the past decade, and this chapter covers several novel aspects of structure-based library design toget ...

The drug discovery process mainly relies on the experimental high-throughput screening of huge compound libraries in their pursuit of new active compounds. However, spiraling research and development costs and unimpressive success rates have driven the development of more rati ...

Multiproperty lead optimization that satisfies multiple biological endpoints remains a challenge in the pursuit of viable drug candidates. Optimization of a given lead compound to one having a desired set of molecular attributes often involves a lengthy iterative process that uti ...

Combinatorial and parallel chemical synthesis technologies are powerful tools in early drug discovery projects. Over the past couple of years an increased emphasis on targeted lead generation libraries and focussed screening libraries in the pharmaceutical industry has driv ...

Optimization of chemical library composition affords more efficient identification of hits from biological screening experiments. The optimization could be achieved through rational selection of reagents used in combinatorial library synthesis. However, with a rapid ad ...

In this chapter we present an application of in silico quantitative structure–activity relationship (QSAR) models to establish a new ligand-based computational approach for generating virtual libraries. The Free–Wilson methodology was applied to extract rules from two data se ...