遗传学实验技术

In the past several years, a host of new technologies have made it possible to visualize single molecules within cells and organisms (Raj et al., Nat Methods 5:877–879, 2008; Par� et al., Curr Biol 19:2037–2042, 2009; Lu and Tsourkas, Nucleic Acids Res 37:e100, 2009; Femino et al., Science 280:585–590, 1998; Ro ...

To distinguish whether differences in gene expression between species or between individuals of the same species are caused by cis-regulatory changes or by distribution differences in trans-regulatory proteins, comparison of species-specific mRNA expression in an F1 hybrid by w ...

Changes in gene expression are an important source of phenotypic differences within and between species. Differences in RNA abundance can be readily quantified between genotypes using a variety of tools, including microarrays, quantitative real-time PCR, cDNA sequencing, and in si ...

Differential gene expression is a key factor driving phenotypic divergence. Determining when and where gene expression has diverged between organisms requires a quantitative method. While large-scale approaches such as microarrays or high-throughput mRNA sequencing can id ...

The Polymerase Chain Reaction (PCR) with its multiple applications in molecular genetic analysis is the cornerstone of modern basic and applied biomedical research. This chapter focuses on the inverse PCR technique that has been used widely over the last two decades in genotyping and chro ...

Rapid amplification of cDNA ends (RACE) is a widely used PCR-based method to identify the 5′ and 3′ ends of cDNA transcripts from partial cDNAs. While conceptually simple, this method often requires substantial optimization before accurate end identification is achieved. This is due in part to ...

Degenerate primers are mixtures of similar oligonucleotides that are used in a PCR, so-called degenerate PCR, to amplify unknown DNA sequences, typically coding sequences of genes. Degenerate primers are designed based on sequence data of related and already sequenced gene homologs. T ...

Next-generation sequencing technologies are revolutionizing the field of evolutionary biology, opening the possibility for genetic analysis at scales not previously possible. Research in population genetics, quantitative trait mapping, comparative genomics, and phy ...

New technologies for DNA sequencing have made it feasible to determine the genome sequence of any organism of interest. This sequence is the resource required to create tools for downstream studies, including DNA microarrays. A number of vendors can produce DNA microarrays containing cu ...

Until recently, gene arrays could only be printed on two types of supports: nylon membranes or glass slides. Nylon membrane-based arrays allow researchers to analyze hundreds of genes in a single experiment using standard laboratory equipment. However, the density of genes that can be inclu ...

A number of articles have been published describing methods to produce fluorescent probes from RNA (or DNA) samples. These methods are conceptually similar. Broadly speaking, they involve some or all of the following procedures: template amplification, template transcription with ...

Highly parallel hybridization of nucleic acids on glass slides has successfully been applied to measure RNA and DNA abundances in Escherichia coli (1–4). In this chapter, we summarize our experience in working with E. coli DNA microarrays accumulated over a 4-yr period. Typically, we printed a ...

DNA microarrays have been used extensively in recent years to study mRNA expression profiles of different cell types under various growth conditions. These steady-state mRNA profiles provide a wealth of information about cellular functions and responses. However, they do not necess ...

A cDNA microarray consists of hundreds or thousands of polymerase chain reaction (PCR)-amplified cDNAs spotted onto a glass microscope slide, in a high-density pattern of rows and columns (1). cDNA microarrays were first used widely to quantify gene expression across hundreds or thousan ...

A critical part of understanding the mechanism and logic of cellular regulatory networks is understanding where enzymes and their regulatory proteins interact with the genome in vivo. From this, we can determine the genomic features that specify protein binding and simultaneously id ...

Statistical considerations are frequently to the fore in the analysis of microarray data, as researchers sift through massive amounts of data and adjust for various sources of variability in order to identify the important genes among the many that are measured. This chapter summarizes so ...

The in vivo analysis of DNA-protein interactions and chromatin structure can provide several kinds of critical information regarding regulation of gene expression and gene function. For example, DNA sequences spanned by nuclease-hypersensitive sites or bound by transcription ...

Southwestern blotting was first described by Bowen et al. (1) and was used to identify DNA-binding proteins that specifically interact with a chosen DNA fragment in a sequence-specific manner. In this technique, mixtures of proteins such as crude nuclear extracts or partially purified pre ...

The ordered and regular packaging of eukaryotic DNA within the chromatin complex allows the efficient utilization of this substrate for nuclear processes such as DNA replication, transcription, recombination, and repair (1,2). Thus, an understanding of the organization of protein ...

The interaction of cell-type-specific or inducible transcription factors with regulatory DNA sequences in gene promoters or enhancers is a pivotal step in genetic reprograming during cell proliferation and differentiation and in response to extracellular stimuli. The study of ...