化学生物学实验技术

The storage of yeast artificial chromosome (YAC) libraries in ordered microtiter plates required a new approach to screening for clones containing specific DNA sequences. Screening libraries of some 60,000 clones by hybridization to filters prepared from individual 96-well micr ...

Yeast artificial chromosome (YAC) libraries stored in microtiter plates are available for screening as either complex PCR pools or hybridization filters generated from YACs gridded at high densities (see Chapter 57). Different libraries may be available as either PCR pools, hybridi ...

The development of the yeast artificial chromosome (YAC) cloning system (1) and the construction of YAC libraries with large numbers of genome equivalents (2–6) provided a major impetus to mammalian genome mapping. These technical advances enabled the mapping of megabase-sized chrom ...

The introduction of yeast artificial chromosomes (YACs) as cloning vectors in 1987 has significantly advanced the analysis of complex genomes (1). The capability of cloning large DNA (100–2000 kb) as YACs has accelerated the construction of physical maps and contig building (a contiguo ...

Cosmid libraries still are an important tool for cloning large genomic regions. Although other Escherichia coli-based vector systems like bacteriophage PI, bacterial artificial chromosomes (BACs), and PI-based artificial chromosomes (PACs) allow the cloning of longer inser ...

The complexity of the genome of a particular organism or the relative abundance of a particular mRNA, within the cell type from which a cDNA library was constructed, affects the ability with which one can isolate a gene or cDNA clone of interest. With respect to genomic libraries, the number of clones nee ...

If an unknown protein is purified and available in relatively small amounts, it is possible to determine the sequences of short internal peptides (1). In order to determine the whole sequence of the protein by cDNA cloning, one of the peptides of perhaps five to seven amino acids may be reverse translat ...

Probably the most commonly used method to screen a cDNA library is hybridization to a labeled DNA probe. This probe may be a single-stranded oligonucleotide or a double-stranded cDNA or polymerase chain reaction (PCR) product. The DNA may be either radioactively or nonradioactively label ...

Much of our current understanding of the molecular details of the activity and interactions of proteins has stemmed from the ability to isolate cDNA encoding these proteins. Computer-based analysis of deduced amino acid sequence allows predictions to be made about their structure and fu ...

The last 10 years have witnessed a growing interest in fungal molecular biology and, in a number of systems, both DNA and RNA technologies have been applied to address various aspects of fungal biology. Consequently, considerable progress has been made in the use of molecular markers, i.e., restri ...

The phage λgt11 system has become increasingly popular for expression of cDNAs or genomic DNAs either in phage plaques or in bacteria lysogenized with recombinant phages (1,2). It offers the advantages of high cloning efficiency, high-level expression, the relative stability of β-gala ...

The genome of the filamentous bacteriophage M13 comprises a single-stranded circular DNA molecule, designated the (+) strand, which is converted into a double-stranded replicative form (RF) on infection of Escherichia coli. Cloning vectors based on M13 RF DNA facilitate the production ...

A variety of methods have been developed and modified to facilitate the cloning of polymerase chain reaction (PCR) products directly into plasmid. However, there are limitations in practice, including the low frequency of correct clones, the expense of commercially available cloning ...

Colony hybridization is a procedure that allows the detection of cells containing nucleic acid sequences of interest (1). In this method, microbial colonies grown on, or transferred to, a supporting membrane are lysed and their nucleic acids denatured to single strands and fixed in place on the ...

Some polymerase chain reaction (PCR) applications such as gene detection or typing (1,2) require little purified DNA and may be performed with crude bacterial extracts. Many methods have been described for this procedure (Chapter 6). Some of them are straightforward and consist of simply bo ...

Plasmids may be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. Many of these are rapid, small-scale “minipreps” that may be used effectively for plasmid analysis and further manipulation. The me ...

The ever-expanding identification of new gene family members in recent years has depended in large part on the use of the polymerase chain reaction (PCR) technique. Typically PCR products may be cloned into a vector by cohesive- or blunt-end ligation. However, both cohesive- and blunt-end liga ...

Subtraction-hybridization cDNA libraries (1–4) are libraries enriched for sequences representing mRNAs whose expression in one biological source (e.g., tissues, cell lines) is different from a second source. Single-stranded cDNAs from both sources are allowed to hybridize so that ...

Mammalian chromosomes are of the order of 12–60 times the size of that of Escherichia coli (4 � 103 kilobase pairs ) (1). The choice of method used when purifying DNA from mammalian cells may be dictated by the use to which the product will be put as it will influence the average size of the material purified. For exam ...

Many methods have been described for isolating DNA from prokaryotic cells. The choice of method depends on the degree of purity of the DNA required for the analysis to be performed. Some DNA analyses (e.g., those using restriction enzymes) require DNA of high purity in relatively large amounts. This ...