遗传学实验技术

Since the invention of dideoxy DNA sequencing (1), the genomes of a number of organisms have been either partially or completely sequenced, including two draft sequences of the human genome (2,3). This offers new opportunities to investigate genetic diversity within and between species. B ...

Over the last few years, association mapping of disease genes has developed into one of the most dynamic research areas of human genetics. It focuses on identifying functional polymorphisms that predispose to complex diseases. Population-based approaches are concerned with exploi ...

In this chapter we lay out some background information about gene mapping for human complex traits. The chapter covers issues such as study design, high-throughput genotyping technologies, statistical analysis, and others. Many of the materials are based on and related to our recent exper ...

Fine-scale mapping methods have been developed to localize functional polymorphisms within large candidate regions identified from previous linkage and/or association studies. Population-based association fine-mapping methods utilize linkage disequilibrium of ...

Imprinted genes in mammals are expressed exclusively from one of the parental alleles (1-6). This is regulated by parental-allele-specific CpG methylation. For example, H19 is methylated exclusively on the paternal allele, which is repressed, and is expressed exclusively from the mat ...

The advantages of using large genomic clones in the analysis of imprinted genes is described in Chapter 4 with particular reference to yeast artificial chromosomes (YACs). These contain on average 500-600 kb of DNA but can be much larger (1 Mb). YACS are propagated in yeast and are therefore amenable ...

F1 hybrids resulting from intercrosses of inbred strains have provided an invaluable tool for the study of imprinting. The hybrids can be used to analyze parent-of-origin differences in expression of any gene, provided sequence differences exist between the two parental alleles. Meth ...

The ribonuclease protection assay (RPA) is a sensitive technique for the analysis of total cellular RNA. It involves generating a specific antisense riboprobe, hybridizing the probe to total RNA, removing unprotected RNA by RNases, and finally isolating and analyzing the protected RNA ...

The seminal work of McGrath and Solter (1) and independently of Surani et al. (2) in 1984 established the fundamental principle of nuclear nonequivalency; that is, chromosomes of both paternal and maternal origin are required for development to term in mammals. This was achieved through the cre ...

Single-strand conformation polymorphism (SSCP) analysis is a sensitive mutation detection system that has been widely used in the field of medical genetics (1,2). In this method, PCR products are denatured to become single-stranded, and separated by gel electrophoresis under nonden ...

Chemical Cleavage of Mismatch (CCM) is one of the methods of choice for mutation research and diagnosis of inherited diseases, as it is capable of detecting 100% of single-base mismatches (1). The scientific background of CCM stems from the initial study of sequencing technique (2) in conjuncti ...

DNA sequencing, while relatively laborious, is the gold standard in mutation detection and single nucleotide polymorphism (SNP) discovery. The most widely used approach is direct DNA sequencing of polymerase chain reaction (PCR) products with dye-terminator chemistry analyzed ...

Single-nucleotide substitutions represent the largest source of diversity in the human genome. Some of these variations have been directly linked to human disease, though the vast majority are neutral. Even neutral variations are important because they provide guideposts in the pre ...

Both the quantity and the distribution of variations in DNA sequence are the product of fundamental biological forces: random genetic drift, demography, population history, recombination, spatial heterogeneity of mutation rates, and various forms of selection. In humans, single b ...

The chief attribute of the fluorogenic 5′ nuclease assay is that it is completely homogeneous. After mixing the sample and reaction components, the assay is run in a closed tube format with no postpolymerase chain reaction (PCR) processing steps. Results are obtained by simply measuring the fl ...

Most nonviral gene transfection vectors deliver transfecting DNA into cells through the endocytic pathway (1,2). Poor escape from endocytic vesicles in many cases constitutes a major barrier for delivery of a functional gene, since the endocytosed transfecting DNA is unable to reach the ...

Retroviral vectors derived from murine leukemia retrovirus (MuLV) have been widely used for efficient gene transfer to achieve long-term expression of a chosen therapeutic gene in mammLian cells (1). Disadvantages of this vector are the instability and low viral titers generated from p ...

Gene gun technology provides a useful means for direct transfer of DNA or RNA constructs that can result in transgenic protein expression from gene expression vectors (1–8). The system has been applied to a broad spectrum of experimental studies on transgenic research, gene therapy approac ...

Recombinant retroviruses are among several virus vectors currently being tested in clinical trials for purposes of gene therapy. As the number of clinical studies increases, accurate quantitation of retrovirus stocks and comparisons between different laboratories and clini ...

Retroviral vectors are optimal gene transfer vehicles for gene therapy, since they integrate into the target cell genome and therefore can provide permanent expression of a therapeutic gene. This is a particularly desired feature in gene therapy of monogenic inherited diseases, in whi ...