遗传学实验技术

Isolation of a full-length gene on the basis of a limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically nucleic ...

Since the first report on cDNA cloning in 1972 (1), this technology has been developed into a powerful and universal tool in the isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. But the conventional methods of cDNA cloning require much effort to generate a li ...

The polymerase chain reaction (PCR) is used for selective amplification of DNA fragments from both prokaryotes and eukaryotes (1–3). The only requirement for amplification is that the sequence of the extremities of the DNA fragment to be amplified be known (4). This places a limitation on the use of ...

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 superfamilies are co ...

Biological systems are often influenced by molecules that are neither present in vast quantities or easily purified to homogeneity from other cellular constituents. The development of simple, efficient molecular cloning systems coupled with the relative ease of DNA sequence dete ...

One of the most important factors affecting the quality of polymerase chain reaction (PCR) is the choice of primers. Several rules should be observed when designing primers and, in general, the more DNA sequence information available, the better the chance of finding an “ideal” primer pair. For ...

Differential (+/-) first-strand cDNA screening methods identify clones corresponding to mRNAs that are expressed at a higher level in one of a pair of phenotypically different cells. This approach is limited by the fact that screening of libraries with labeled first-strand cDNAs synthes ...

The “megaprimer” method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing ...

Site-directed mutagenesis permits modification of the functional characteristics of specific proteins and the characterization of regulatory DNA elements. Since the amplifying primers used in the polymerase chain reaction (PCR) are incorporated into the product, PCR can be us ...

Although the polymerase chain reaction (PCR) (1,2) is invaluable for the cloning and manipulation of existing DNA sequences, PCR also makes it possible to create new DNA fragments consisting of a nucleic acid sequence that is specified entirely by the investigator. In this chapter we describe a ...

Gene Splicing by Overlap Extension (gene SOEing) provides a powerful method of recombining sequences without depending on restriction sites or ligase, and a simple, generally applicable way of using polymerase chain reaction (PCR) to perform site-directed mutagenesis in vitro. This ...

The capacity to recombine and modify DNA are underpinnings of the recombinant DNA revolution. The polymerase chain reaction (PCR) (1,2) provides a rapid means for the site-directed mutagenesis of DNA and for the recombination of DNA (1-9). Recently, two methods have been introduced that perm ...

The transfection of expressible DNA into cell lines has allowed studies of gene expression and interaction that are often very difficult or impossible using the endogenous genes. The importance of this method is highlighted by the observation that, in a recent issue of Cell, 10 out of 14 articles r ...

A few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (PCR) amplification procedure to identify and orient a plasmid insert (1,2). By combining this procedure with one in which asymmetrically ampli ...

The melding of a technique for repeated rounds of DNA synthesis with the discovery of a thermostable DNA polymerase has given scientists the very powerful technique known as polymerase chain reaction (PCR). PCR is based on three simple steps required for any DNA synthesis reaction: (1) denatur ...

The reverse transcription-polymerase chain reaction (RT-PCR) is a powerful tool when studying gene expression in a limited number of cells. RT-PCR was first described by Veres et al. (1) and numerous accounts have since followed. Classic studies of gene expression have utilized Northern bl ...

Messenger RNAs of higher eucaryotes are usually modified posttran-scriptionally to contain 5′ caps and nucleotide methylation, 3′ polyadenylation, and one or more internal splices to remove introns and join exon segments into the mature protein-coding sequences. With the abiliti ...

Quantitative measurement of specific mRNA species is of major importance for approaching many fundamental questions in biology. Until now, quantitation of gene expression has usually been done by Northern blotting, but this procedure is relatively insensitive, requiring micro ...

Reverse transcription of RNA followed by the polymerase chain reaction (RT-PCR or RNA-PCR) is an extraordinarily sensitive method to detect as few as 1–100 copies of a specific RNA (1-3). However, we and others have found that false positives caused by contamination with minute quantities of DNA ( ...

Chromatin structure analysis at single-nucleotide resolution can be done by genomic sequencing, and, recently, several techniques have been developed (1) that give improved specificity and sensitivity over the original method of Church and Gilbert (2). The most sensitive method us ...