基因表达差异显示实验

The newly discovered phenomenon of RNA interference (RNAi) offers the dual facility of selective viral gene silencing coupled with ease of tailoring to meet genetic variation within the viral genome. Such promise identifies RNAi as an exciting new approach to treat virus-induced disea ...

The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patt ...

Whole-genome profiling using DNA arrays has led to tremendous advances in our understanding of cell biology. It has had similar success when applied to large viral genomes, such as the herpesviruses. Unfortunately, most DNA arrays still require specialized and expensive resources and, g ...

Processivity factors associate with DNA polymerases, enabling them to incorporate thousands of nucleotides without dissociating from the template. The processivity factors encoded by each of the herpesviruses are ideal targets for specifically blocking viral replicatio ...

A commonly used method for labeling nucleic acid probes nonradioactively is to introduce a small, relatively inert molecule, such as a hapten or biotin. Such molecules are generally coupled to a nucleoside triphosphate, such as dUTP, via a linker group. The modified nucleotide is then incorpo ...

Site-directed mutagenesis is a powerful technique by which specific changes can be generated in a target DNA molecule for either structure-function investigations or for creating and deleting endonuclease restriction sites. There have been several oligonucleotide-direct ...

Oligonucleotide-directed mutagenesis can be used to introduce specific alterations in cloned DNA, including base-pair substitutions, insertions and deletions. It is an important procedure in studies of gene expression and protein structure/function relationships. A var ...

The development of the polymerase chain reaction (PCR) has allowed the rapid isolation of DNA sequences utilizing the hybridization of two oligonucleotide primers and subsequent amplification of the intervening sequences by Taq polymerase. There are many applications of this tec ...

The dideoxy chain-termination method of DNA sequence analysis involves the synthesis of a DNA strand by enzymatic extension from a specific primer using a DNA polymerase (1). Several different enzymes are available for this purpose, each having different qualities and properties.

Isolating a clone from a cDNA or genomic library often involves screening the library by several rounds of plating and filter hybridization. This is not only laborious and time-consuming, but also is prone to artifacts, such as false positives commonly encountered in filter hybridization. ...

Following transformation of a ligation reaction into competent E. coli cells, successful subclones are conventionally identified by two methods. The first involves preparing “mini-prep” plasmid DNA from a number of colonies and then identifying the desired recombinant plasmid on ...

Radioactively labeled polymerase chain reaction (PCR) products are being used in an increasing number of molecular biology research techniques. Among these are PCR-based polymorphism assays, such as linkage analysis (1) and detecting allelic loss in cancer cells (2). Other uses of rad ...

The polymerase chain reaction (PCR) has revolutionized the way that molecular biologists approach the manipulation of nucleic acids through its ability to amplify specific DNA sequences (1–3). It has numerous applications (4), many of which require the cloning of amplified DNA products ...

As more and more genes are cloned and sequenced, it is apparent that nearly all genes are related to other genes. Similar genes are grouped into families. Examples of gene families include the collagen, globin, and myosin gene families. There are also gene superfamilies. Gene super-families are co ...

Many techniques are currently available that allow the isolation of DNA (1–7) or RNA (8–23), but such methods allow only the purification of one type of nucleic acid at the expense of the other. Frequently, when cellular material is limiting, it is desirable to isolate both RNA and DNA from the same source. S ...

There are probably as many “miniprep” recipes as there are laboratories doing molecular biology. The following protocol is derived from the alkaline lysis recipe originally described by Birnboim and Doly (1), which was slightly modified in the 1982 edition of Moleculur Cloning: A Laborat ...

In recent years several techniques have been described for the introduction of DNA molecules into Escherichia coli. These are based on the findings of Mandel and Higa (1), who demonstrated that incubation of cells with naked bacteriophage DNA in cold calcium chloride resulted in uptake of vir ...

Southern blotting is a well-known technique (1). DNA is cleaved with restriction enzymes (Chapter 2) to produce fragments that are fractionated according to their size in an agarose gel (Chapter 3). The DNA is then partially cleaved by depurination (to facilitate the transfer of larger DNA frag ...

The RNase protection assay is based on the resistance of RNA:RNA hybrids to single-strand specific RNases, after annealing to a complementary 32P-labeled probe in solution. It can be used to map the ends of RNA molecules or exon-intron boundaries. It also provides an attractive and highly sensi ...

Small-molecule drugs are a cost-effective way to treat and prevent disease. A study by the Slone Institute published in 2002 estimated that over 50% of the adult population in the United States used at least one pharmaceutical drug during the preceding week. The positive impact of small-molecule ...