遗传学实验技术

DNA vaccination represents a powerful new approach for the elicitation of long-lived protective immunity against a broad range of protein antigens (1,2). In this approach, the vaccine is a plasmid DNA vector that encodes a foreign protein to be targeted for the induction of humoral or cellular r ...

Bacterial DNA, but not vertebrate DNA, causes direct stimulation of several components of the vertebrate immune system. This activation is due to the presence of unmethylated CpG dinucleotides (1), which are present at the expected frequency in bacterial DNA, but are underrepresented (“ ...

Many infectious diseases of global impact are caused by parasites. This includes diseases with protozoan etiology, such as malaria, African sleeping sickness, Chagas disease, toxoplasmosis, and amoebiasis, as well as diseases caused by metazoa, such as river blindness, schistosom ...

Yeast have proven to be a valuable and flexible host for cloning and manipulating large fragments of foreign genetic material, providing valuable reagents for the study of gene function and regulation in a variety of applications. However, this approach requires a familiarity with basic p ...

Large-scale genome mapping and sequencing projects nowadays rely on robotic systems for yeast artificial chromosome (YAC) library processing, handling, and analysis. The advantages are obvious. Robots are fast, less labor intense, and the data reproducibility is high. However, the ...

Restriction enzyme analysis of yeast artificial chromosome (YAC) clones in combination with Southern blotting and polymerase chain reaction (PCR) is necessary to establish the integrity of the DNA contained within the YAC clone, as well as obtain information on the integrity of the clon ...

The free availability of multiple genomic sequences represents one of the greatest advances in biology of the new millennium, and promises to revolutionize our ability to determine and treat the causes of human disease. This chapter highlights a number of basic, freely available, and user- ...

Yeast artificial chromosomes (YACs) are useful cloning vectors with the capacity to carry large DNA inserts. The largest barrier using such large DNA molecules in transformation experiments has been their physical instability in a solution. We developed a new method for transforming y ...

The ability to efficiently and accurately modify genomic DNA through targeted and tissue-specific mutations is an important goal in animal transgenesis. Here we describe how to exploit two systems of homologous recombination, from yeast and bacteria, to engineer yeast artificial ch ...

The development of yeast artificial chromosome (YAC) technology permits cloning of DNA segments that can be thousands of kilobases in size. This facilitates techniques such as the generation of transgenic animals as a YAC clone can, in most cases, carry an entire gene with all its regulatory ele ...

Transformation-associated recombination (TAR) cloning allows selective isolation of full-size genes and genomic loci as circular yeast artificial chromosomes in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, ch ...

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 ...