生物化学技术

The amplification refractory mutation system (ARMS) is a simple and rapid method of detecting point mutations, restriction fragment length polymorphisms, and small deletions or insertions of a DNA sequence. The method was first described by Newton et al. (1) for the analysis of single nucle ...

The Sanger dideoxy-nucleotide sequencing method has been simplified by a number of methodological improvements, such as the use of the polymerase chain reaction (PCR) technique for generating DNA templates in sufficient quantities, followed by affinity-capture techniques for ...

RNA dot hybridizations were first described by Kafatos et al. (1). These hybridizations allow rapid analysis of mRNA expression and are particularly useful in the initial characterization of clones derived from differentially expressed genes. Where accurate quantification of tr ...

Denaturing-gradient gel electrophoresis (DGGE) detects DNA sequence differences. Thus, it can be used to screen for point mutations or other types of mutation prior to DNA sequence analysis. The technique, first described by Fischer and Lerman (1), entails electrophoresis of DNA fragme ...

Screening for mutations prior to sequencing can reduce the time and costs of identifying mutations. When the DNA sequence is known, the technique of detecting mutations as single-stranded conformational polymorphisms (SSCP) is a convenient method of screening for possible mutatio ...

Chemical cleavage of mismatch (CCM) is one of the mismatch cleavage methods, all of which allows scanning of 1–2 kb lengths in one test (reviewed in ref. 1). Heteroduplexes are formed between wild-type and mutant DNA (or RNA) and reacted with hydroxylamine, which modifies matched or unmatched C bases ...

DNA sequence changes within a gene results either in polymorphism or mutation causing different diseases. Some of these polymorphisms that occur with a high frequency within the population can be a useful tool for gene tracking for a given disease. Such investigations have initially been do ...

Because polymorphisms are noted by the presence or absence of amplification products from a single allele, the random amplified polymorphic DNA (RAPD) technique tends to provide only dominant markers. Individuals containing two copies of one allele are not distinguished by amplifi ...

DNA fragments may be separated by gel electrophoresis in a gel composed of agarose. This allows DNA fragments to be resolved on the basis of their molecular weight. The percentage of agarose used depends on the size of fragments to be resolved. In general a 0.8–1% gel may be used for effective separation of DNA ...

A major advance in physical mapping of the human genome was the development of yeast artificial chromosome (YAC) vectors (1). This has enabled the cloning of pieces of DNA several hundred kilobases in length (2). The availability of such large cloned genomic DNA fragments means that by ordering a se ...

The technology of exon trapping, sometimes called exon amplification, strives to exploit the phenomenon of mRNA splicing to discover genes directly from genomic DNA. There are three distinct exon trapping methodologies that differ simply in the genomic target of interest. The original ...

Changes in cell behavior are driven by changes in gene expression. Thus, in order to understand the mechanisms regulating cell behavior, one has to identify and characterize differentially expressed genes. Standard methods currently used to isolate differentially expressed genes ...

The ability to cleave DNA at specific sites is one of the cornerstones of today’s methods of DNA manipulation. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. These enzymes can be purchased from ...

Conventional polymerase chain reaction (PCR) enables reliable amplification of 3–4 kb of DNA (1) while attempts at optimization has enabled 15.6 kb of λ DNA to be amplified (2). The maximum amplifiable length of PCR is limited by the low fidelity of the Thermus aquaticus (Taq) DNA polymerase (3), the mo ...

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 ...

Often, studies require the polymerase chain reaction (PCR) amplification of several nucleic acid targets. Examples include the amplification of several exons of the same gene for mutation detection (e.g., p53 or dystrophin) (1–4), the amplification of several different genomes for inf ...

Rapid amplification of cDNA ends (RACE) amplifies either terminal of a cDNA, even where they have unknown sequence (1,2). RACE only requires knowledge of a short sequence within the mRNA of interest. It is often used for cloning the remainder of incomplete cDNAs.

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 ...

As more and more genes are cloned and sequenced, it is apparent that nearly all genes aie related to other genes. Similar genes are grouped into families, such as the collagen and globin gene families. There are also gene superfamilies. Gene superfamilies are composed of genes that have areas of high ho ...

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 ...