Family-based association methods are useful because they offer improved matching of controls to cases, with the result that they are not susceptible to confounding by population stratification. They also allow analysis of parent-of-origin effects and maternal–fetal interacti ...
Candidate gene association studies aim to establish or characterise association between the genetic �variation occurring within a specific gene or locus and a phenotype. If the phenotype is quantitative, then the effect size is often measured as the difference between the genotype spe ...
In this chapter, mutation (specifically single-nucleotide polymorphisms, SNPs) and recombination will be covered in more detail, and the concepts of genotype and haplotype will be reviewed. Linkage disequilibrium (LD) describes the strength of a relationship between alleles at d ...
Preimplantation development is a complicated process, which involves many genes. We have investigated the expression patterns of 17 developmentally important genes and isoforms in early mouse embryos as well as in single cells of the mouse embryo. The comparison is an excellent example ...
A long reverse transcription polymerase chain reaction (LRP) protocol is described for the amplification of large RNA sequences. The amplification of near full-length hepatitis C virus (HCV) genome from serum samples is used as an example to detail each step in LRP procedure, including pri ...
Cloning of infectious and pathogenic herpesvirus genomes in a bacterial artificial chromosome (BAC) vector greatly facilitates genetic manipulation of their genomes. BAC-based mutagenesis strategies of viruses can advance our understanding of the viral gene functions and d ...
Campylobacterand Helicobacterspecies are important pathogens in man and animals. The study of their virulence and physiology has been difficult due to the lack of tractable genetic tools, since many of the techniques established in Escherichia coliand related species were found to be ...
In vitro selection coupled with directed evolution represents a powerful method for generating nucleic acids and proteins with desired functional properties. Creating high-quality libraries of random sequences is an important step in this process as it allows variants of individ ...
This chapter describes the use of degenerate primers for PCR amplification of orthologous DNA from related species. While several methods for designing degenerate primers have been described, an important consideration is to base the design on a short region of highly conserved amino ac ...
Functional analysis of a protein of interest, by generation of functional alterations in a target protein, often requires the performance of site-directed mutagenesis within the gene sequence. These manipulations are usually performed using “cut and paste” techniques, combined w ...
A wide variety of random- and site-directed mutagenesis techniques have been developed to investigate the structure–function relationship in proteins and intergenic regions like promoter sequences. Similar techniques can be employed to optimize protein properties like ena ...
Deciphering protein function is a major challenge in modern biology and continues to remain at the frontier of investigations into the molecular basis of cell behavior. With the explosion in our bioinformatics knowledge base and the now widespread use of associated software tools and dat ...
When designing a mutagenesis experiment, it is often crucial to estimate the stability change of proteins induced by mutations (Δ DG). Despite the recent advances in computational methods, it is still challenging to estimate D DG quickly and accurately. We recently developed the Eris proto ...
The complexity of human illnesses often extends beyond a single mutation in one gene. Mutations at other loci may act synergistically to affect the penetrance and severity of the associated clinical manifestations. Discovering the additional loci that contribute to an illness is a chall ...
The insertion of peptide loops into the polypeptide chain of proteins at surface-exposed regions is an attractive avenue to modify the protein’s properties or to evolve new functionalities. The strategy of peptide loop insertion has, for example, been used to create new binding sites in prot ...
A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant l ...
Three major techniques have been employed for broad-range in vitro mutagenesis of Brucella species. Shotgun approaches capable of generating large libraries of randomly inserted transposon mutants include Tn5, mariner (Himar1), and mini-Tn5 signature-tagged mutagenesis. A ...
Varicella-zoster virus (VZV) causes both varicella (chicken pox) and herpes zoster (shingles). As a member of the human herpesvirus family, VZV contains a large 125-kb DNA genome, encoding 70 unique open reading frames (ORFs). The genetic study of VZV has been hindered by the large size of viral geno ...
Mutagenesis by the overlap extension PCR has become a standard method of creating mutations including substitutions, insertions, and deletions. Nonetheless, the established overlap PCR mutagenesis is limited in many respects. In particular, it has been difficult to make an inserti ...
Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. ...
