Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger me ...
Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for acc ...
The revolution in molecular techniques over the last 30 years detracted from many traditional cytological techniques for examining basic biological problems. One of these casualties is the preparation of karyotypes and analysis of chromosomal structure, behaviour, and variat ...
With the rapid expansion of whole-genome sequencing and other genomic studies in nonmodel �organisms, there is a growing demand for robust and user-friendly methods for estimating eukaryotic genome sizes across a broad range of taxa. Propidium iodide (PI) staining with flow cytometry is a ...
A variety of applications require the creation of custom-designed plasmids, including transgenic reporters, heterologous gene fusions, and phenotypic rescue plasmids. These plasmids are created traditionally using restriction digests and in vitro ligation reactions, but ...
Cis-regulatory sequences control when, where, and how much genes are transcribed. A better understanding on these elements is a fundamental keystone to better understand development, cell differentiation, and morphogenesis. Several methods based on in silico analysis or ChIP-seq ...
Identifying mechanisms of molecular adaptation can provide important insights into the process of phenotypic evolution, but it can be exceedingly difficult to quantify the phenotypic effects of specific mutational changes. To verify the adaptive significance of genetically b ...
Elucidating the molecular bases by which phenotypic traits have evolved provides a glimpse into the past, allowing the characterization of genetic changes that cumulatively contribute to evolutionary innovations. Historically, much of the experimental attention has been fo ...
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 ...
