遗传学实验技术

By determining an organism’s DNA sequence, researchers can obtain critical information about its development and physiology, its taxonomic relations, and its susceptibility to disease. There are three distinct methods of acquiring DNA sequence information: sequence-speci ...

Since its earliest days, the history of human genetics has been checkered with actual, perceived, and potential abuses in the application of its scientific concepts to research or clinical endeavors. Aside from obvious cases of scientific fraud and continuing controversies over natur ...

High-density polynucleotide probe arrays provide a massively parallel approach to genetic sequence analysis that is having a major impact on biomedical research and clinical diagnostics (1). These arrays are comprised of large sets of nucleic acid probe sequences immobilized in de ...

The ongoing progress in establishing a reference sequence as part of the Human Genome Project (1) has revealed a new challenge: the large-scale identification and detection of intraspecies sequence variations, either between individuals or populations. The information drawn from ...

DNA microarray technologies have been developed as a high-throughput means to study transcriptional regulation (for a recent review, see ref. 1). Large numbers of DNA samples (either complete cDNAs or ESTs) are immobilized as very high-density arrays on solid surfaces, typically glass. M ...

The creation of microspot arrays of bioactive materials has been vigorously pursued by a number of research organizations and commercial companies in the past decade (1,2). In addition to the high density and/or small area achieved by using microspot arrays, increased sensitivity is poss ...

Miniaturization and high-throughput parallel analysis are new concepts entering modern molecular biology. An exciting example of such technology originally developed by physicists and applied to biology is the microchip. The semiconductor industry manufactures silica c ...

Statistics is often thought to concern only the analysis of observational or experimental data. However, experimental design is one of the oldest subfields of statistics. The founder of modern statistics, R. A. Fisher, noted that “statistical procedure and experimental design are only t ...

In this chapter we discuss the problem of identifying differentially expressed genes from a set of microarray experiments. Statistically speaking, this task falls under the heading of “multiple hypothesis testing.” In other words, we must perform hypothesis tests on all genes simulta ...

Clustering is the task of organizing a set of objects into meaningful groups. These groups can be disjoint, overlapping, or organized in some hierarchical fashion. The key element of clustering is the notion that the discovered groups are meaningful. This definition is intentionally vague, ...

Clustering is one of the most commonly used tools in the analysis of gene expression data (1,2). The usage in grouping genes is based on the premise that coexpression is a result of coregulation. It is often used as a preliminary step in extracting gene networks and inference of gene function (3,4). Cluste ...

DNA microarrays represent a powerful technology offering unprecedented scope for discovery (1). However, the ability to measure, in parallel, the gene expression patterns for thousands of genes represents both the strength and a key weakness of microarrays. One of the central challeng ...

B lymphocytes that infiltrate solid tumors, known as tumor-infiltrating lymphocyte B-cells (TIL-B-cells), are present in human tumors from different histological types (1,2) and tumor-specific antibodies can be detected in many cancer patients. For instance, anti-idiotypic an ...

The intracellular expression of recombinant single-chain Fv (scFv) molecules—termed intrabodies—has potential advantages for therapeutic use (1,2). These single polypeptide chains are made of one heavy-chain variable region (VH) linked through a flexible spacer, usually a re ...

Cell-sorting and mRNA amplification techniques with RT-PCR have been combined to study the gene expression of cytokines (1–3), receptors (4–6) and differentiation antigens of purified hematopoietic cells (7–9). Nevertheless, a major drawback of these techniques is the difficulty in ...

Reverse-transcriptase-polymerase chain reaction (RT-PCR) is a powerful and sensitive tool for RNA detection. This technique is particularly valuable in cases where the amount of an RNA species present is so minute that traditional RNA analysis methodologies such as Northern blot or R ...

The rate-limiting steps in the positional cloning of disease genes are the identification of discrete genes within a large genomic region and the analysis of mutations in these genes. Direct selection is an expression-based gene identification technique that can rapidly identify cDN ...

There are currently a number of techniques available for the isolation of novel coding sequences from cloned genomic fragments. The most recent methodologies include exon trapping (1), direct selection (2,3), and screening of cDNA libraries with whole radiolabeled yeast artificial c ...

Since the advent of polymerase chain reaction (PCR), it has proven possible to clone complete genes, or parts of these, without having to construct the usually obligatory cDNA library. PCR cloning, however, is not without its own problems and limitations, such as the fidelity of the cloned sequence ...

Temporal and spatial expression of the 100,000 different genes in the genome of a mammal is a highly regulated process that determines the normal development of an organism. This assumption is based on the fact that only a fraction of these genes, in the range of 10,000–15,000, are expressed in any cell ty ...