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The reverse transcription – polymerase chain reaction (RT-PCR) is a sensitive technique for the quantification of steady-state mRNA levels, particularly in samples with limited quantities of extracted RNA, or for analysis of low level transcripts. The procedure amplifies defined m ...

Mutagens, clastogens, and aneugens cause increased expression of the human GADD45a gene. This has been exploited in the GreenScreen HC genotoxicity assay in which the gene’s expression is linked to the expression of green fluorescent protein (GFP). The host for the reporter construct is the h ...

The detection and characterisation of DNA adducts can provide mechanistic information on mode of action for genotoxic chemicals and in this context is vital for human risk assessments. Adducts are measured extensively in biomonitoring studies to examine exposure to environmental, ...

The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the “comet-like” appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in ...

The in vitro micronucleus test detects genotoxic damage in interphase cells. The in vitro micronucleus test provides an alterative to the chromosome aberration test, and because the in vitro micronucleus test examines cells at interphase, the assessment of micronuclei can be scored fa ...

Chromosome aberration analysis has been the basis of one of core tests in genetic toxicology since guidelines were first established (DHSS (1981) Guidelines for the Testing of Chemicals for Mutagenicity. Prepared by the Committee on Mutagenicity of Chemcials in Food, Consumer Produc ...

32P-postlabelling is a technique originally described by Kurt Randerath and colleagues for the sensitive detection of damage produced in DNA by reactive chemicals or genotoxins. The procedure essentially entails the enzymatic digestion of DNA to nucleoside 3′-monophosphates w ...

Human apurinic/apyrimidinic endonuclease-1 (APE-1) is essential for base excision repair and plays a major role in DNA repair and maintaining genomic stability. Cancer cells treated with conventional DNA-damaging agents develop resistance due in part to upregulation of enzymes i ...

Recent interest in inactive kinase conformations has generated the need to develop new biochemical tools to study them. Here, we describe the use of a fluorescent probe that selectively and potently binds to a specific inactive conformation of protein kinases. This allows for the thermody ...

Several protein kinases have been characterized in a specific inactive form called the DFG-out conformation. Unlike the active conformation which is conserved in all kinases, the inactive DFG-out conformation appears to be accessible to only certain kinases. This inactive conform ...

There is growing interest in exploring allosteric sites on proteins for drug discovery. At the center of the regulation of many protein kinases from the AGC family there is an allosteric site termed “PIF-pocket.” The regulated binding of a C-terminal region of the kinase to the PIF-pocket, within t ...

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), the most common cancer in AIDS patients. All herpesviruses express a conserved dimeric serine protease that is required for generating infectious virions and is therefore of pharmac ...

Accumulating work over the past decade has shown that peptide aptamer screening represents a valid strategy for inhibitor identification that can be applied to a variety of different targets. Because of the screening method in cells and the highly combinatorial libraries available, th ...

Virtual ligand screening is a powerful technique to identify potential hits of targets and to increase hit rates. Here, we describe how we used this technique combined with NMR 15N HSQC experiments to obtain small molecules that bind to the PDZ domain of Dvl targeting the Wnt signaling pathway.

Small molecule docking and virtual screening of candidate compounds have become an integral part of drug discovery pipelines, complementing and streamlining experimental efforts in that regard. In this chapter, we describe specific software packages and protocols that can be used ...

RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA nanoparticles can be fabricated with a level of simplicity characteristic of DNA while possessing versatile tertiary structure and catalytic function similar to that of proteins. A large v ...

MicroRNAs (miRNAs) are highly conserved small RNAs which regulate gene expression primarily through base pairing to the 3′ untranslated region of target messenger RNA (mRNA), leading to mRNA degradation or translation inhibition depending on the complementarity between the miRNA ...

The discovery of oncogenes and tumor suppressors as a driver of cancer development has triggered the development of target-specific small molecule anticancer compounds. As exemplified by Imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia-associated BCR/ ...

This chapter provides an overview of the Bayesian approach to data analysis, modeling, and statistical decision making. The topics covered go from basic concepts and definitions (random variables, Bayes’ rule, prior distributions) to various models of general use in biology (hierarc ...

The maximum likelihood method is a popular statistical inferential procedure widely used in many areas to obtain the estimates of the unknown parameters of a population of interest. This chapter gives a brief description of the important concepts underlying the maximum likelihood met ...