遗传学实验技术

Microbes, which constitute a major fraction of the total biomass, are the main source of biodiversity on our Planet and play an essential role in maintaining global processes, which ultimately regulate the functioning of the Biosphere. Recent emergence of “metagenomics” allows for the an ...

Numerous gene-specific PCR methods have been developed for the cultivation-independent discovery of novel genes from complex environmental DNA samples. The recovery of full-length genes is, however, technically challenging. Here, we present an efficient and relatively simple ...

A DNA microarray-based approach is described for screening metagenomic libraries for the presence of selected genes. The protocol is exemplified for the identification of flavin-binding, blue-light-sensitive biological photoreceptors (BL), based on a homology search in alre ...

We consider a gene finding method that is specifically designed to work on metagenome sequences. The method can handle short metagenome sequences with in-frame stop codons as well as frame shifts. It delivers gene predictions for a set of metagenome sequences, which may be individual reads or a co ...

Sequence-based screening tools of a metagenome library can expedite metagenome researches considering tremendous metagenome diversities. Several critical disadvantages of activity-based screening of metagenome libraries could be overcome by sequence-based scre ...

Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still u ...

The vast majority of the Earth’s biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. In the last few year ...

The choice of an expression system for the meta-genomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for the meta-genomic gene libraries. However, the use of E. c ...

We describe how wide host-range cloning vectors can lead to more flexible and effective procedures to isolate novel genes by screening metagenomic libraries in a range of bacterial hosts, not just the conventionally used Escherichia coli. We give examples of various wide host-range plasm ...

This detailed protocol describes an approach for combining DNA stable-isotope probing-based enrichment, multiple displacement amplification (MDA), and metagenomics. Together, these three methodologies enable selective access to the genomes of uncultivated organis ...

One of the few available systems for gene expression in (hyper)thermophilic Archaea is the virus-based shuttle vector pMJ05 for Sulfolobus solfataricus. Although it is still not fully developed and there are some difficulties arising from the large size of the vector (20 kb), it has successf ...

The functional expression of environmental genes in a particular host bacterium is hampered by various limitations including inefficient transcription of target genes as well as improper assembly of the corresponding enzymes. Therefore, the identification of novel enzymes fr ...

The functional overproduction of proteins is an essential step required for providing enzymes and proteins for practical applications. Accordingly, the expression system used is important, e.g., cis-acting elements including promoters for the expression of recombinant prot ...

The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this cha ...

Due to its excellent transfectability, the human embryonic kidney (HEK) 293 cell line is widely used as an in vitro model system for transfection experiments. Particle bombardment, or biolistics technology, provides a physical transfection approach that can deliver transgene mate ...

Particle bombardment is an efficient method for virus inoculation of intact plants. This technique enables inoculation with full-length infectious clone cDNA, PCR products, virus from sap or virus preparation, and in vitro viral transcripts. The inoculation of some phloem-limited ...

Non-vacuum gene guns such as HandyGun are flexible tools for bombardment of targets of varying size. Construction of HandyGun is simpler and cheaper than vacuum gene guns and will be described here. The conditions for maximal transient transformation efficiency of plant cells with plasm ...

Particle bombardment/biolistic delivery is a very popular method of genetic transformation of diverse targets including cells and intact tissues. Delivery of DNA through particle bombardment is genotype and species independent, nevertheless, an efficient protocol for larg ...

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as A ...

Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle �bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only rece ...