基因表达差异显示实验

Modern standardized methodologies, described in detail in the previous chapters of this book, have enabled the software-automated design of optimized DNA construction protocols. This chapter describes how to design (combinatorial) scar-less DNA assembly protocols using the w ...

SLiCE (Seamless Ligation Cloning Extract) is a novel cloning method that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of tra ...

In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression s ...

Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need ...

We developed a simple method (Simple Cloning) for subcloning one, two, or three DNA fragments into any location of a targeted vector without the need for restriction enzyme, ligase, exonuclease, or recombinase. This cloning technology can be applied to a few common Escherichia coli hosts (e.g., B ...

PCR is a common method to produce desired DNA fragments from templates. The oligonucleotide primers used for PCR must contain annealing sequences that are usually 20–30 nucleotides long and identical to a part of template DNA. However, primers often contain additional sequences at their 5′ e ...

The immense amount of gene sequences available nowadays allows scientist to screen broadly for extraordinary proteins. Reliable cloning tools that allow the parallel processing of many targets are vital for the success of this strategy. The FX cloning procedure detailed here is such a str ...

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction–ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces na ...

Many of our behavioral and physiological processes display daily oscillations that are under the control of the circadian clock. The core molecular clock network is present in both the brain and peripheral tissues and is composed of a complex series of interlocking transcriptional/tra ...

The sirtuins are NAD+-dependent, multifunctional lysine deacylases that play key roles in cellular homeostasis. They are increasingly being found to target a variety of substrates including acetyl-, butyryl-, malonyl-, and succinyl-lysines. Early assays for measuring sirtuin ac ...

The identification of lysine-acetylated proteins and deacetylase substrates has primarily relied on protein immune-affinity techniques with antibodies that recognize acetylated lysine residues (Kac antibodies). While these antibody-based techniques are continu ...

Mass spectrometry (MS) allows for the large-scale identification of multiple peptide analytes in complex mixtures. However, the low abundance of acetylated peptides in the overall mixture requires an enrichment step. After enrichment, the resulting acetylated peptides of inter ...

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is one of the in vivo metabolic labeling methods widely used for dynamic analysis of protein modifications. Here, we describe a general approach to applying SILAC, in combination with affinity enrichment of acetyllysine pepti ...

Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs) (Rush et al., Nat Biotechnol 23:94–101, 2005). Here, we describe a general approach for immuno ...

Most of the sirtuins’ nuclear substrates identified so far are histones or other chromatin-associated proteins and, thus, it is of special relevance the development of good biochemical techniques to analyze the biology of these proteins in the context of chromatin. Here, we describe sever ...

The sirtuins are a family of NAD+-dependent deacylases with important effects on aging, cancer, and metabolism. Sirtuins exert their biological effects by catalyzing deacetylation and/or deacylation reactions in which Acyl groups are removed from lysine residues of specific prot ...

Drosophila melanogaster is one of the most widely used genetic model systems in biology. The ease of working in an invertebrate model system allows the design and execution of many experiments that would be infeasible in a vertebrate model. Although the strength of the fly as a model system lies prim ...

The nematode Caenorhabditis elegans (C. elegans) has four Sir2 paralogs, sir-2.1, sir-2.2, sir-2.3, and sir-2.4. Thus far, most of the research tools to study worm sirtuins have been developed for sir-2.1, due to its homology to yeast SIR2 and human SIRT1. Here, we have compiled a listing of the currently ava ...

Originally discovered as a transcriptional silencing protein, SIR2 was later linked to yeast replicative aging and the rest was history. Sir2p is now known to be a member of a class of protein deacetylases with a unique enzymatic activity coupling the deacetylation event to NAD+ hydrolysis. Wh ...

Over the past 15 years, the number of papers published on sirtuins has exploded. The initial link between sirtuins and aging comes from studies in yeast, in which it was shown that the life span of yeast mother cells (replicative aging) was proportional to the SIR2 gene dosage. Subsequent studies have s ...