遗传学实验技术

1. Introduction One of the most important attributes of the polymerase chain reaction (PCR) is its exquisite sensitivity. However, this high sensitivity has also given PCR its main weakness, namely, its tendency to produce false-positive results due to exogenous contamination (1,2). Con ...

A major limitation of solution phase polymerase chain reaction (PCR) is the inability to visualize and localize amplified product within cellular and tissue specimens. In situ hybrrdization (ISH) does permit localization of specific nucleic acid sequences at the individual cell le ...

Although the polymerase chain reaction (PCR) is generally believed to be capable of detecting a single target DNA molecule, it is important to note that there are essentially two different types of “low copy number PCR.” The first type involves the amplification of an isolated DNA target, such as si ...

Many of the anticancer chemotherapeutic agents in current clinical use are known to interact strongly with DNA (e.g., Adriamycin, cis-platinum, melphalan, mitomycin C, and mitoxantrone). Although the exact mechanism of action remains unresolved in most cases, the apparent role of DNA has ...

The synthesis in vitro of DNA-binding proteins, like Myb, offers many advantages over other expression systems, particularly if large amounts of protein are not required and protein modification, including phosphorylation, is preferable. It is a relatively rapid procedure to subcl ...

Work with chloroplast RNA polymerase from various plant species has established the presence of transcription factors that either act on the DNA template or on other polypeptides of the transcription complex. The first example was a 26-kDa polypeptide from maize chloroplasts, referr ...

Much of our current understanding of the mechanisms of cellular protein targeting and membrane translocation of proteins derives from studies on mitochondria. The mitochondrial import process has been studied both in whole cells (including extensive use of mutants, particularly ...

Clostridial DNA is known to contain a high content (1). Cloning of several clostridial neurotoxin genes revealed that such genes contain about 73% in the coding region and up to 85% in the noncoding regions (2–4). The bias for -rich codons may impose difficulties in expressing portions of such genes in E ...

Wheat-germ extract provides a reliable, convenient, and easy-to-use system to initiate translation from a wide variety of viral, prokaryotic, and eukaryotic mRNAs and produce full-length polypeptide products (1). Translation reactions in vitro may be directed by either mRNA isolat ...

Eukaryotic RNA polymerase II requires a set of protein factors for the accurate initiation of gene transcription (1). Whole-cell and nuclear extracts containing these components have been prepared from cultured cells and used to execute faithful transcription initiation on the DNA t ...

The large size, resilience, and high translational capacity of the fullgrown Xenopus oocyte has made it a widely used system for the translation of microinjected natural and synthetic mRNAs. The injected and endogenous mRNAs compete for the oocyte translational machinery in a process th ...

Plastids, the characteristic organelles of plant cells, contain their own DNA and a complete apparatus for transcription and translation (1). Although this is principally similar to what is known for mitochondria, the details of the plastid gene expression system appear to be more complex, ...

Steroid hormones, such as estrogen, progesterone, androgens, glucocorticoids, and mineralocorticoids, are well-known regulators of the expression of specific gene networks in higher eukaryotes (1–3). The hormonal action is mediated through specific intracellular recept ...

The chain termination DNA sequencing procedure introduced by Sanger and coworkers in 1977 (1) meant a revolutionary achievement in fast, simple, and accurate deciphering of DNA fragments. Long stretches of this nucleic acid could from then on be sequenced within a relatively short time. The ...

For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in E. coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques like ultracentrifugati ...

Historically, DNA sequencing by primed synthesis and chain terminators has been performed using the Klenow fragment of DNA polymerase 1(1. This enzyme was derived from E. coli. DNA Pol I which, in addition to polymerase activity, has both 3′–5′ and 5′–3′ exonuclease activities.

The dideoxy chain termination DNA sequencing procedure introduced in 1977 (1) has the advantage of being fast, simple to perform, and very accurate. Therefore, it became the method of choice to obtain several hundred bases of sequence information per reaction. The procedure is based on the enzy ...

Chemical (1) and enzymatic (2) methods for determining DNA sequences have revolutionized the techniques of molecular genetics and subsequently our understanding of the gene. The use of singlestranded Ml3 bacteriophage (3–5), in conjunction with Sanger’s dideoxy chain-terminat ...

A major problem of the directed deletion of double-stranded DNA using Exonuclease III (Chapter 8) is the necessity to find two enzyme sites to open up the insert for digestion and to protect the primer site. The “Cyclone Sequencing” technique of Dale, McClare, and Houchins (1) overcomes this problem ...

Exonuclease III (Exo III) will digest double-stranded DNA in a 3′ to 5′ direction if the DNA is blunt-ended or possesses a 5′ overhang. It will not digest if there is a 3′ overhang of three or more bases, or if the 3’ end has had thiophosphate-containing bases incorporated into it. In order to generate a set of insert dele ...