遗传学实验技术

Polymerase chain reaction (PCR) optimization and troubleshooting can consume considerable energy and resources because of the finicky and often unpredictable nature of the reactions. Small variations in any of the many variables in a given reaction can have a pronounced effect on the re ...

Long polymerase chain reaction (PCR)(1 5), specifically, XL PCR (Extra-Long Polymerase Chain Reaction), has enabled amplification of expanded trinucleotide repeats of the neuromuscular disease myotonic dystrophy (6) a 9-kb HIV-1 provirus from primary isolate DNA (7), 24.2-kb fragm ...

Although Thermus aquaticus (Taq) and Thermus thermophilus (Tth) DNA polymerases have the ability to reverse transcribe RNA to complementary DNA (cDNA) and subsequently amplify the target cDNA, they are not usually the first choices for reverse transcription-polymerase chain rea ...

Many common molecular biology techniques including polymerase chain reaction (PCR) (1), Southern blotting (2), comparative genomic hybridization (3), and in situ hybridization (4) have been adapted for use with paraffin-embedded tissue (PET). PCR-amplified products from PET can be ...

Polymerase chain reaction (PCR) has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs (1-5). We have adapted the concept of long PCR technology to reverse-transcription (RT) PCR (6). Here, we describe the paramet ...

The amplification of GC-rich templates by any PCR method is usually a difficult task and despite the development of modified methods and conditions, this type of amplification still remains a specific case approach. Problems usually observed with GC-rich DNA are constraint of template am ...

A successful cloning of polymerase chain reaction (PCR)-derived DNA fragment is a key step for further analysis of the amplified DNAs, though it is often a difficult task. Many cloning methods have been established; various commercial cloning kits are also available. These methods can be sepa ...

Microsatellites, also referred to as short tandem repeats (STR) or simple sequence repeats (SSR), are highly polymorphic and abundant sequences dispersed throughout most eukaryotic nuclear genomes (1-3). In recent years, microsatellites have been used for linkage map constructi ...

Numerous techniques have been developed for the cloning of polymerase chain reaction (PCR) products. These include the incorporation of restriction enzyme sites into the PCR primers (1), blunt-end cloning (2,3), TA cloning (4,5), ligation-independent cloning (LIC) (6-11), and in vivo clo ...

The polymerase chain reaction (PCR) technique has proved to be a powerful tool for rapid amplification of DNA fragments of interest during cloning. Insertion of PCR products into suitable vectors in order to construct plasmids for protein expression, or to create chimeric genes or to study in ...

The expression of cloned genes in prokaryotic or eukaryotic host cells provides the means not only for the study of gene function but also for the production of substantial amounts of protein and nonprotein molecules for commercial and investigational use. In the case of proteins, strategies ...

Efficient strategies for the production of recombinant proteins are gaining increasing importance, as more applications that require high amounts of high-quality proteins reach the market. Higher production efficiencies and, consequently, lower costs of the final product are ...

Recombinant protein production has become an essential tool for providing the necessary amounts of a protein of interest to either research or therapy. The target proteins are not in every case soluble and/or correctly folded. That is why different production parameters, such as host, cult ...

The use of the improved BAC system for cloning genomic DNA and library constructions is described. This system retains all the advantages of the original BACs but, in addition, permits, on command, amplification of the BAC plasmids and cloned DNA. This system consists of (1) plasmid pBAC/oriV cont ...

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expres ...

Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recom ...

Moderately halophilic bacteria of the family Halomonadaceae (Halomonas, Chromohalobacter, and Zymobacter) have promising applications in biotechnology as a source of compatible solutes (stabilizers of biomolecules and cells), salt-tolerant enzymes, biosurfactan ...

Moderately halophilic bacteria (MHB) of the genera Halomonas and Chromohalobacter have been used as hosts for the expression of heterologous proteins of biotechnological interest, thus expanding their potential to be used as cell factories for various applications. This chapter ...

This review reports some results from our laboratory on the setting up of a psychrophilic expression system for the homologous/heterologous protein production in cold-adapted bacteria by using natural plasmids as cloning vectors. By screening some Antarctic bacteria for the pres ...

The extensive variety of plasmid-based expression systems in E. coli resulted from the fact that there is no single strategy for achieving maximal expression of every cloned gene. Although a number of strategies have been implemented to deal with problems associated to gene transcription ...