遗传学实验技术

Mapping of transcribed mRNA and protein coding sequences is the initial step in functional characterization of genomic sequences. There are several techniques to identify transcribed sequences; however, Northern blot analysis remains a standard method for detection and quant ...

High-content analysis methods provide the opportunity to interrogate specific cellular end points in living cells. When coupled with high-throughput RNA interference (ht-RNAi) loss of function screens, high-content analyses are a powerful discovery tool for the identificat ...

Over the last decade, genetic studies have identified numerous associations between single nucleotide polymorphism (SNP) alleles in the human genome and important human diseases. Unfortunately, extending these initial associative findings to identification of the true cau ...

This chapter reviews statistical issues related to gene association studies. The goal is to review various aspects of study design and analysis for individuals who do not have an extensive statistical background. We will review statistical issues as they relate to both genome-wide and can ...

Genome-wide association studies (GWAS), in which thousands of single-nucleotide polymorphisms (SNPs) spanning the genome are genotyped in individuals who are phenotypically well characterized, �currently represent the most popular strategy for identifying gene regions ...

Depending on the scope of the research project, categories of single-nucleotide polymorphism (SNP) genotyping experiments range from low to medium to high throughput, with each approach differing widely in cost, platform, and efficiency. Medium-throughput genotyping is genera ...

Pooling genomic DNA samples within clinical classes of disease for use in whole-genome single nucleotide polymorphism (SNP) genotyping allows for rapid and inexpensive genome-wide association studies (GWAS). We describe here a general outline for combining hundreds of genomic D ...

A new noninvasive method for the detection of DNA damage using mid-to high-frequency ultrasound (10–60 MHz) has been developed. Ultrasound imaging and quantitative analysis methods are used to detect cell death occurring in response to anticancer therapies in cell samples in vitro, in rat b ...

Extensive DNA fragmentation that generates a multitude of DNA double-strand breaks (DSBs) is a hallmark of apoptosis. A widely used approach to identify apoptotic cells relies on labeling DSBs in situ with fluorochromes. Flow or image cytometry is then used to detect and quantify apoptotic c ...

This chapter describes a technique in which indirect immunofluorescence is applied to visualize the process of nucleotide excision repair (NER) at the site of locally induced damage in DNA. UV-irradiation of cells through an isopore polycarbonate membrane filter generates cyclob ...

DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) permits simultaneous and selective labeling of single- and double-strand DNA breaks in individual cells, either in the whole genome or within specific DNA sequences. In this technique, cells are embedded into ag ...

The comet assay is a simple and sensitive method for measuring DNA damage. Cells are embedded in agarose on a microscope slide, lysed, and electrophoresed; the presence of strand breaks allows the DNA to migrate, giving the appearance of a comet tail, the percentage of DNA in the tail reflecting the break ...

Recently, the concept of apoptotic cell elimination was expanded and programed cell death is no longer viewed as an individual cellular event. The complete description of the apoptotic process now includes two phases: the self-driven cell disassembly and the externally-controlled e ...

The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selec ...

The in situ ligation (ISL) methodology detects apoptotic cells by the presence of characteristic DNA double-strand breaks. A labeled double-stranded probe is ligated to the double-strand breaks in situ on tissue sections. Like the popular TUNEL assay, ISL detects cells in apoptosis based ...

The native T7 DNA polymerase is a fast and highly processive enzyme that can be used for in situ detection of apoptosis and various types of DNA breaks. The technique is quick and simple, and was shown to label earlier stages of apoptosis compared to the terminal transferase technique. The in situ labeli ...

A method for the localization of DNA strand breaks at the ultrastructural level is presented. The technique involves the use of terminal deoxynucleotidyl transferase and labeled dUTP. Incorporation of labeled nucleotides is visualized through colloidal gold labeling. Cells und ...

Oxidative cell damage causes disruption of DNA via formation of 8-hydroxy-2′-deoxyguanosine and can trigger apoptotic cell death. The cells damaged by oxidative stress can either become apoptotic, or recover. Therefore, it is helpful to employ a parallel assay that would confirm whether ...

Apoptosis, or programmed cell death, plays an important role in normal development and homeostasis of adult tissues. Apoptosis has also been linked to many disease states, including cancer. One of the biochemical hallmarks of apoptosis is the generation of free 3′-hydroxyl termini on DNA via ...

Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of f luorochrome-labeled inhibitors of ...