遗传学实验技术

Large, multicomponent complexes mediate many stages of eukaryotic gene expression, from transcription to translation. Despite their size, these complexes and their precursors can often be resolved and analyzed by native gel electrophoresis. Although other techniques exist f ...

Inverse PCR (IPCR) was designed for amplifying anonymous flanking genomic DNA regions (1 2). The technique involves the digestion of source DNA, circulation of restriction fragments, and amplification using oligonucleotides that prime the DNA synthesis directed away from the core r ...

Isolation of a full-length gene and analysis of expression profiling are fundamental and challenging in the current molecular biology. A great deal of effort is needed to detect unknown gene sequences by screening cDNA or genomic libraries by nucleic acid or protein probes. As the complete ge ...

Because of its versatility, the Tn5 transposon has become a powerful tool in the classical genetic studies of Gram-negative bacteria. The Tn5 transposon is functional in a broad range of Gram-negative bacteria and transposes at a high frequency with low specificity of insertion (1,2), allow ...

Current and recent efforts to determine the genomic DNA sequence for numerous organisms (e.g., Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Arabidopsis thaliana, Zea mays, Caenorhabditis elegans, Mus musculus, Homo sapiens, Schizosaccharomyces pombe, D ...

Large insert libraries are critical in genome-related research. Bacterial artificial chromosome (BAC) libraries are widely used in plant, animal, and human research; the ends of BAC clones are used as probes for chromosome walking and to confirm overlapping of contigs, as well as RFLP marke ...

Traditionally, libraries are screened with different probes to isolate target genes or sequences. These probes can be a particular sequence such as a cDNA, a polymerase chain reaction (PCR) product, or a genomic fragment (1). An oligonucleotide can be a probe if no closely related cDNAs or gene clo ...

The hunt for missing sequence data, whether it be in pursuit of full-length clones or for promoter sequences, can be laborious and expensive. Indeed, protracted efforts to find missing sequences by library screening can be fraught with the frustration of foraging through libraries that may l ...

Conventional procedures to isolate a gene belonging to an ortholog family usually imply the use or the construction of double-stranded cDNA libraries derived from a specific mRNA source of interest (cells or tissues) (1). The double-stranded DNA library plated on various membranes is scr ...

The advent of the polymerase chain reaction (PCR) has greatly facilitated the isolation and characterization of clones from both cDNA and genomic libraries (1-3). Given the complexity of the genome of a particular organism or the relative abundance of a particular mRNA, within the cell type fr ...

Now that the draft sequence of the human genome is available (1,2), cDNA cloning based on its sequence from a library is no longer an experimental goal, but a starting point and a routine laboratory practice. Hybridization screening with either radio-labeled or nonradio-labeled probes had been ...

The simple sublibrary method described in this chapter allows the detection and rapid isolation of rare clones from bacteriophage λ libraries. The method is based on the ability of polymerase chain reaction (PCR) to detect clones present in a library at very low frequencies. Clones present at f ...

The bacterial artificial chromosome (BAC) (1) is now the most widely utilized vector system to construct large-insert genomic libraries for genome analysis. It has the following advantages over another vector system, the yeast artificial chromosome (YAC) (2): ease of DNA purification, a ...

As the genome research continues to grow and genomic information avalanches at the speed of light, biological research labs are facing continuous effort to redefine the scale and scope of their research. Large-scale research applications such as high-throughput sequencing and cDNA mi ...

The construction and sequencing of DNA libraries, such as genomic libraries and cDNA libraries, is an important and powerful approach to understand the biology at the most fundamental level, that of the DNA. The high-throughput DNA sequencing approach has also been used in novel gene discove ...

A promising approach towards high-throughput genotyping of single nucleotide polymorphisms (SNPs) is to use arrays of immobilized oligonucleotides in miniaturized assays (1,2). A significant advantage of performing the assays in microarray formats is that the costs of genotyp ...

Quantitative analysis of pooled DNA samples is today generally recognized as a promising approach to determine allele frequencies of single nucleotide polymorphism (SNP) markers. The use of pooling will increase the genotyping throughput in population genetic studies or associ ...

The primer extension assay with fluorescence polarization (FP) is a genotyping method that combines the specificity of nucleotide incorporation by DNA polymerase and the sensitivity of fluorescence polarization. We named the assay Template-directed Dye-terminator Incorp ...

Allele-specific (AS) PCR amplification (1–3) has been used in combination with gel based detection to genotype-specific polymorphisms. Until recently a major drawback of this method was that it was labor-intensive and without high-throughput instrumentation (4). The single nucle ...

In the basic Invader assay, two synthetic oligonucleotides, the invasive and signal probes, anneal in tandem to the target strand to form the overlapping complex shown schematically as primary reaction in Fig. 1. The signal probe is designed to have two regions: a target-specific region that is c ...