遗传学实验技术

The ability to isolate genes that encode specific proteins has been fundamental to the revolution in molecular and cell biology over the past two decades. The protein-encoding versions of genes are isolated from cDNA libraries. A cDNA library is constructed by isolating mRNA from the cell or ti ...

The construction of high-quality, full-length cDNA libraries is still the ideal method for cloning and studying mRNA populations. However, a significant problem with conventional cDNA library construction is the requirement for microgram quantities of poly A+ RNA at the start of the pro ...

The basic reverse-transcriptase-polymerase chain reaction (RT-PCR) technique is used routinely and widely in most biomedical research laboratories. The fundamental considerations for such basics as primer design, good laboratory set up and practice, and techniques for perf ...

Reverse-transcriptase-polymerase chain reaction has become one of the most widely applied techniques in biomedical research. The ease with which the technique permits specific mRNA to be detected and quantified has been a major asset in the molecular investigation of disease patho ...

Every researcher who generates new sequence data will have a curiosity and a need to know if his or her sequence is the same or similar to one that has been identified before, if it is likely to contain part of a gene, and the possible function of any such encoded protein. As the Human Genome Project accelerates tow ...

Direct sequencing of PCR products using the dideoxy chain termination procedure developed by Sanger et al. (1) is now the most commonly used method for defining specific mutations. The main benefits of this method lie in its ease of use and this has been enhanced in recent years by the introduction of fl ...

DNA sequencing methods were first developed more than 20 years ago with the publication of two approaches to sequencing methodology that became known as Sanger sequencing (1), based on enzymatic synthesis from a single-stranded DNA template with chain termination using dideoxynucl ...

For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in Escherichia coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques such as ultrac ...

The sequencing of DNA has undergone rapid improvement since the introduction of the chain-termination DNA sequencing method (1) and the construction of convenient single-stranded DNA cloning vectors, such as the bacteriophage M13 cloning vectors and their derivatives (2,3). The ch ...

The dideoxy chain termination DNA sequencing procedure introduced in 1977 (1) has the advantage of being fast, simple to perform, and very accurate. Therefore, it became the method of choice to obtain several hundred bases of sequence information per reaction. The procedure is based on the enzy ...

The direct sequencing of an area of DNA using the product of a polymerase chain reaction (PCR) as template is a very powerful technique. It is straightforward and fast and therefore the method of choice in situations such as the analysis of mutations. The reliability of such analysis depends greatly ...

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1–5). The CFTR gene spans about 250 kb at the genomic level (6). After transcription, a transcript of about 6.1 kb is obtained containing up to 27 exons (2,7). Several alternatively sp ...

When the Human Genome Mapping Project began in earnest, the allocation of large funding and the opening of dedicated research facilities promised much for the rapid improvement of DNA sequencing methods and, as a consequence, sequencing rates and throughput. Today, several years later, so ...

Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanis ...

The integron includes a site-specific recombination system capable of integrating and expressing genes contained in structures called mobile gene cassettes. Integrons were originally identified on mobile elements from pathogenic bacteria and were found to be a major reservoir ...

Although horizontal gene transfer (HGT) is often considered as a disruptive force in reconstructing organismal phylogeny, it can also be a valuable phylogenetic tool. A gene in the net of life is often horizontally transferred to the ancestor of a major lineage. If the gene is retained in the recipi ...

A universal Tree of Life has been a longstanding goal of the biosciences. The most common Tree of Life, based on the small subunit rRNA gene, may or may not represent the phylogenetic history of microorganisms. The horizontal transfer of genes from one taxon to another provides a means by which each gene may ...

Horizontal gene transfer (HGT) is a driving force in the evolution of metabolic pathways, allowing novel enzymatic functions that provide a selective advantage to be rapidly incorporated into an organism’s physiology. Here, the role of two HGT events in the evolution of methanogenesis is d ...

The detection of horizontal gene transfer (HGT) events has become an increasingly important issue in recent years. Here we discuss a simple theoretical analysis based on the in silico artificial addition of known foreign genes from different prokaryotic groups into the genome of Escheri ...

This chapter discusses the pros and cons of the existing computational methods for the detection of horizontal (or lateral) gene transfer and highlights the genome-wide studies utilizing these methods. The impact of horizontal gene transfer (HGT) on prokaryote genome evolution is dis ...