遗传学实验技术

MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary miRNAs (pri- ...

RNA-induced silencing complexes (RISCs) are multiple-turnover entities that direct many rounds of site-specific target mRNA cleavage (1). A principal RISC component in all eukaryotes is a member of the Argonaute (AGO) protein family (4). AGO contains the conserved PAZ and PIWI domains, and ...

Conditional gene targeting is often a useful approach to elucidate the in vivo function of a gene. We use this approach to investigate the biological role of the RNA interference (RNAi) pathway in mammals. In addition, the RNAi machinery in mammalian cells can be exploited for gene knock-down expe ...

I describe the use of RNAhybrid, a program that predicts multiple potential binding sites of microRNAs (miRNAs) in large target RNAs. The core algorithm finds the energetically most favorable hybridization sites of a miRNA in a large potential target RNA. Intramolecular hybridization ...

In this chapter, we review evidence that at least three different types of microRNA (miRNA)-messenger RNA (mRNA) target interactions exist in mammals: short seeds, long seeds, and “perfect” hits (allowing G:U matches). Because new types of miRNAs are still being discovered, this list may not yet be ...

MicroRNAs (miRNAs) are small, nonprotein-coding RNAs that regulate gene expression. Although hundreds of human miRNA genes have been discovered, the functions of most of these are unknown. Computational predictions indicate that miRNAs, which account for at least 1% of human protein- ...

If an unknown protem is purified and available in relatively small amounts, it is possible to determine the sequences of short internal peptides (1). In order to determine the whole sequence of the protein by cDNA cloning, one of the peptides of perhaps five to seven amino acids may be reverse translated ...

Transformation of Escherichia coli was first described by Mandel and Higa (1), who reported that E. coli cells, after treatment with calcium chloride, can take up bacteriophage λ DNA and produce viable phage particles. The conditions for the transfer of exogenous DNA into E. coli have been examin ...

The method described in this chapter has been used in the molecular cloning of transcription factors and other factors with DNA-binding activity toward specific double-stranded DNA sequences. The protocol is based on the method of Singh et al. (1) and shares feature with the immunological ap ...

Cycle sequencing of polymerase chain reaction (PCR) products has become the method of choice for sequencing known genomic DNA fragments. When fluorescent DNA sequencers are used, this requires the incorporation of a fluorescent dye into the cycle sequencing product either by using lab ...

Oligonucleotide arrays (also known as DNA chips, gene chips, or genosensor chips) are emerging as a powerful research tool in nucleic acid sequence analysis. Several technical challenges remain to be solved, however, before oligonucleotide arrays can reach their full potential and be im ...

This chapter focuses on sequencing by hybridization (SBH), an advanced DNA sequencing technique first proposed in 1987 (1). SBH procedures determine DNA sequence information by screening DNA oligomers (typically 7-to 11-mers) for their ability to hybridize with target DNA. The set of ove ...

Antisense oligonucleotides (AONs) are synthetic deoxyribonucleic acids, typically between 15–25 nucleotides, that can bind to complementary sites in a mRNA and inhibit translation. AONs show promise as therapeutics to many diseases caused by abnormal or unwanted expression of a g ...

Over the last few decades, typing for the human leukocyte antigen (HLA) has become critical for bone marrow transplants. Research has also indicated that individuals with certain allelic genotypes may be at a higher risk for developing diseases affecting the immune system, and therefore ra ...

Previous chapters have discussed in detail how to prepare, print and hybridize DNA arrays on various surfaces. Once hybridization is completed, the next step is to scan in the glass, gel, or plastic slides with a specialized scanner to obtain digital images of the results of the experiment. The DNA exp ...

As the Human Genome Project continues toward its goal of characterizing the genome of human and selected model organisms through complete mapping and sequencing of their DNA, unique opportunities are becoming available for studying genetic variation in humans and its relationship w ...

Confocal scanning laser microscopes are best known for making very high-resolution images of small three-dimensional (3D) specimens by recording a series of two-dimensional confocal slices of the specimen at different focus positions. Additionally, confocal microscopes are e ...

In the context of this chapter, biochips are defined as microscale bioanalytical devices that incorporate microfluidic circuitry, highly parallel functionality, or both. Microfluidic circuitry has yielded the lab-on-a-chip concept in which functions such as sample processi ...

Biochips are small platforms with spatially arrayed macromolecules (or pieces thereof) that allow the collection and analysis of large amounts of biological information. The principle of the technology is based on specific molecular recognition interactions between the array ...

It may seem premature to be writing a history of DNA microarrays because this technology is relatively new and clearly has more of a future than a past. However readers could benefit from learning something about the technical basis of DNA microarrays, and younger readers may be curious to know somet ...