The propensity of Taq polymerase to add 3′-A overhangs (1,2) to polymerase chain reaction (PCR)-amplified DNA has made possible a simple method for cloning PCR products into a T-vector (Invitrogen, San Diego, CA) (3–5). Here, we present a related strategy that uses T-linkers to add sequences, such as re ...

Polymerase chain reaction (PCR) mediated through Taq DNA polymerase has become a simple and routine method for cloning, sequencing, and analyzing genetic information from very small amounts of materials (1). Taq DNA polymerase, like some other DNA polymerases, lacks 3′ to 5′ exonuclease ac ...

In the past decade, the primed in situ (PRINS) labeling technique has become an alternative to fluorescence in situ hybridization (ISH) for the localization of nucleic acid sequences in chromosome, cell, and tissue preparations (1–8). The PRINS method is based on the rapid annealing of unlabel ...

The advent of the reverse transcriptase polymerase chain reaction (RT-PCR) technique represents a quantum leap in sensitivity over preceding methods of detecting mRNA transcripts, such as Northern blotting. With the arrival of such sensitive techniques, it has become possible to am ...

In recent years, the development of in situ technologies has made good progress. In situ hybridization (ISH) has become an important tool and has enabled the pathologist to demonstrate infectious pathogens or mRNAs in tissue sections or cytospins without destruction of morphology, thus e ...

Primed in situ amplification (PRINS) is a technique for the visualization of specific sequences, usually repeat sequences, in fixed cell nuclei. When viewed on the microscope, the resulting signals can be seen as spots within nuclei, providing a means to visualize telomeres, centromeric r ...

The polymerase chain reaction (PCR) is an extremely sensitive technique allowing the detection of rare and low copy nucleic acid sequences (up to 1–10 copies in DNA or mRNA extracts from 1 million cells) by solution-phase amplification using specific primer sets and Taq DNA polymerase and visu ...

There are a number of template types that are generally recognized as being difficult to sequence. These can include sequences with a high guanine-cytosine (G/C) content, sequences that are very rich in adenine/thymine (A/T), sequences with a marked secondary structure, or large regions of ho ...

Strategies for cloning polymerase chain reaction (PCR) products and performing in vitro site-directed mutagenesis are legion, and the following chapters outline five robust and reliable protocols. Before embarking on such a strategy, however, it is worth considering if it is entirely ...

Microarray-based screening technologies have revealed a larger than expected diversity of gene expression profiles for many cells, tissues, and organisms. The complexity of RNA species, defined by their molecular structure, represents a major new development in biology. RNA not on ...

Many elegant methodologies have been devised to explore RNA-protein as well as RNA–RNA interactions. Although the characterization of messages targeted by a specific RNA-binding protein (RBP) has been accelerated by the application of microarray technologies, reliable methods ...

The study of recombination in Saccharomyces cerevisiae benefits from the availability of assay systems that select for recombinants, allowing the study of spontaneous events that represent natural assaults on the genome. However, the rarity of such spontaneous recombination re ...

Recombination is involved in many important biological processes including DNA repair, gene expression, and generation of genetic diversity. Recombination must be carefully regulated so as to prevent the deleterious consequences that may result from rearrangements between ...

Because of the availability of the complete sequence of the genome of the model plant Arabidopsis and of insertion mutants for most genes in public mutant collections, the elucidation of the particular role of different factors involved in DNA recombination and repair processes, an import ...

Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis. DSBs are also induced by chemicals and radiation. I ...

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites of mammals that were first described over 100 hundred years ago. They have long been the subjects of biological investigation, which has yielded insights into a number of fundamental, as well as novel, cellular proc ...

The goal of understanding the function of all mammalian genes is best accomplished through mutational analyses. Although the sequence of the mouse genome is now available and many genes have been identified, it is not possible to ascribe functions accurately to these genes in silico. Gene tar ...

The yeast DEL assay is a simple, rapid method for measuring the frequency of reversion of a disrupted his3 gene by homologous intrachromosomal recombination. Reversion to histidine prototrophy results in deletion (DEL) of the disrupting sequence. The DEL assay has been used to study the effe ...

Large-scale genomic rearrangements such as DNA deletions play a role in the etiology of cancer. The frequency of DNA deletions can be elevated by exposure to carcinogens or by mutations in genes involved in the maintenance of genomic integrity. The in vivo DNA deletion assay allows a visual detec ...

The ability to make specific genomic alterations is an invaluable tool to researchers who use genetics and biochemistry to study problems in biology. We have investigated some of the parameters governing DNA fragment transplacement in two commonly used strains of Saccharomyces cere ...