遗传学实验技术

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. ...

Massive Mutagenesis� is a proprietary library creation method that enables the fast generation of high-quality genetic libraries. Starting from a single gene on a plasmid and hundreds to thousands of oligonucleotides, a one-step single-strand circular amplification method crea ...

Directed evolution experiments are performed to improve the properties of proteins by creating a library of mutated genes of interest and selecting those genes that encode proteins exhibiting desired properties. Here, we present one of the methods to carry out an evolutionary experime ...

Marker genes, such as gusA, lacZ, and gfp, have been applied comprehensively in biological studies. Directed in vitro evolution provides a powerful tool for modifying genes and for studying gene structure, expression, and function. Here, we describe a strategy for directed in vitro evoluti ...

Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components s ...

Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will increase PCR amplification efficiency as well as isolate the targeted sequence of interest with higher specificity. Many ...

Reverse transcription (RT) is the synthesis of complementary deoxyribonucleic acids (DNA) from single-stranded ribonucleic acid (RNA) templates. This process is catalyzed by the reverse transcriptase enzyme, which is the replicating enzyme of retroviruses. Reverse transc ...

Extraction of high quality RNA is paramount to successful RT-PCR, and here, a method proven optimal for skeletal muscle is described. While this method described is for use with skeletal muscle, it could be suitable for other types of mammalian tissue also. This method describes an approach to extr ...

Here, we report a detailed procedure for the exact quantification of minute amounts of nucleic acids by competitive PCR. This technique entails the co-amplification of a target DNA or cDNA in a biological sample together with a known quantity of a target-specific standard, the competitor, whi ...

We describe a single step reverse transcription polymerase chain reaction protocol that can be used to amplify part of the neuraminidase gene segment (segment 6) from all nine subtypes of influenza A virus. The method has also been applied to amplify gene segment 1 of influenza A, which encodes the b ...