生物化学技术

Plant transformation involves the insertion of “foreign” genetrc material into plant cells and the regeneration of transgenic plants Over the past 15 yr or so, a variety of methods for plant genetic engineering have been described, these fall into two broad categories. Agrobactenum-bas ...

The isolation of high-quality cellular DNA is often a starting point for a variety of molecular-biology techmques These include Southern-blot analysis, PCR amphfication, and genomic-library construction (Fig. 1) It is often necessary to use the DNA from a single preparation for a number of d ...

Recombmant baculovinrus expression systems are now firmly established as one of the tools for producing foreign proteins in high yield wlthin a eukaryotlc environment The evolution of both single- and multiple-transfer vectors and different forms of baculovlral DNA has greatly str ...

The descrrptron of the first retrovtral packaging cell lme in the early 1980s mtroduced the technology by which a wide variety of different genes could be reliably and efficiently transduced into target cells using retrovnal vectors (I). Although the early prototype packaging systems we ...

Bacteriophage-based vectors, such as λ have been useful in creating gene llbrarles and in the expression of proteins However, a number of techniques used in molecular biology require DNA to be ina single-stranded form, such as chain termmatlon sequencing, ollgonucleotlde-directed mu ...

Plasmids, circular molecules of DNA that rephcate autonomously in a bacterial host cell, are widely distributed among prokaryotes They vary in size from a few to several hundred ktlobase pairs Although generally not essential to cellular survival, plasmids can confer a selecttve advan ...

Genes can be identified and cloned by complementanon of mutants, hybrrdtzation of nucleic acids, screening for their products, or systematic sequencing of genetic enttties. The chotce of the strategy to be applied for tsolating a gene of interest has to be taken in a space of parameters mcluding, ...

Genomic DNA libraries are a collection of DNA fragments that together represent the entire (or nearly entire) genome of the mdividual from which the DNA was derived. These fragments are contained within self-rephcating vectors that enable them to be mamtamed and propagated within the cells ...

Messenger RNA (mRNA) 1s a complex mixture of easily degraded molecules representing the processed (splrced, polyadenylated, and capped) transcripts from genes that are active in a given tissue or cell type In the construction of a cDNA library, RNAdependent DNA polymerase (reverse tran ...

The rsolatron of intact, high quality, total cellular RNA is often the starting point for many molecular biologrcal procedures (Fig. 1) There are numerous general methods for the rsolation of total cellular RNA (1–9). There are also many specialrzed methods for the rsolatron of RNA from specific ...

Synthetic oligonucleotides are important in a wide variety of applications ranging from use as hybridization probes (1), primers for DNA sequencing and the polymerase chain reaction (2,3), to utilization as potential therapeutics in antisense and related technology investigat ...

Major uses of oligodeoxyribonucleotides are as hybridization probes, sequencing primers, and, more recently, as primers for the polymerization chain reaction. When a protein sequence or part thereof is known, the construction of oligomer probes and primers is complicated by the codon ...

DNA fragments labeled with 15N have the potential to provide novel insight into base pairing, hydration, drug/nucleic acid, and protein/ nucleic acid interactions. Synthetic routes to a variety of -labeled pyrimidine nucleosides (1–3), purines (4–6), and purine nucleosides (7–13) have b ...

For nucleic acid hybridizations, the choice of nonisotopic label is constrained by hybridization and wash conditions, the sensitivity needed in the assay, and the detection method. Several recent reviews (1–4) have summarized methods of labeling synthetic DNA probes. In spite of signi ...

The attachment of reporter groups, drug derivatives, or chemically reactive species to DNA in a sequence-specific manner has the potential to provide new materials for detailed spectroscopic and structural analyses as well as new classes of DNA therapeutics and diagnostics. Sequence ...

Solid-phase oligodeoxynucleotide synthesis has become a routine procedure in most molecular-biology laboratories. The reagents for the synthesis of unmodified oligomers have been available for several years, and novel commercially available reagents that permit the intr ...

The most common technique to label oligodeoxynucleotides is the enzymatic incorporation of the radioisotope 32Pat the 5′-end. Although this method affords high sensitivity of detection, the use of the radiolabeled probe is precluded in many clinical and diagnostic applications. In ...

Nucleobases with attached side chains have been incorporated into oligodeoxynucleotides (ODNs) to achieve a number of goals. The functionalized side chains have been used to attach reporter groups (protein ligands as well as fluorescent moieties) and metal-chelating groups. Ele ...

A large number of research groups have focused considerable effort toward thermodynamically characterizing melting transitions in nucleic acid molecules. This interest reflects the fact that the resulting thermodynamic data permit one to define the nature of the molecular for ...

The application of mass spectrometry (MS) to the structural analysis of large biopolymers, such as nucleic acids, combines the desirable characteristics of low-level detectability with high selectivity. Current MS techniques are capable of providing mol wt and structurally spec ...