遗传学实验技术

Comparative evolutionary analyses of molecular sequences are solely based on the identities and differences detected between homologous characters. Errors in this homology statement, that is errors in the alignment of the sequences, are likely to lead to errors in the downstream ana ...

Whole-genome alignment (WGA) is the prediction of evolutionary relationships at the nucleotide level between two or more genomes. It combines aspects of both colinear sequence alignment and gene orthology prediction, and is typically more challenging to address than either of these t ...

Genomes can be organised in different ways. Understanding the extent of the diversity of genome organisation, the processes that create it, and its consequences is particularly important for two key reasons. Firstly, it is relevant for our understanding of the genetic basis for the astound ...

Genomic analyses increasingly make use of sophisticated statistical and computational approaches in investigations of genomic function and evolution. Scientists implementing and developing these approaches are often computational scientists, physicists, or math ...

This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a sing ...

In recent years, DNA microarray has become increasingly popular as a tool to investigate global expression patterns compared to differential display RT-PCR. Although differential display RT-PCR can be labour-intensive, it has its own merits over those of DNA microarray. While the latt ...

Microarray gene expression profiling has identified gene signatures or “Indicator” genes predictive of outcome in many cancer types including lymphoma, and more recently pancreatic cancer. This has identified novel and powerful diagnostic and prognostic and generically app ...

Codon saturation is a powerful tool for analyzing protein structure–function relationships and fine-tuning enzyme activity. In this technique, one or more key amino acids are randomized by incorporating degenerate codon(s) into a gene of interest in a polymerase-mediated primer ex ...

Mammalian adaptation to stressful stimuli requires activation of the type 1 corticotropin releasing hormone (CRH) receptor (CRH-R1), a 415 aminoacid protein that belongs to the large superfamily of 7 transmembrane domain receptors that relay signals across cells through activat ...

Recombinant antibody fragments are significant therapeutic and diagnostic reagents. As such, their efficacy depends heavily on their affinities and biophysical properties. Thus, mutagenesis approaches have been extensively applied to recombinant antibodies to improve ...

Many proteins, especially transporters, are thought to undergo large conformational alterations during their functional cycle. Since X-ray crystallography usually gives only the most stable conformation, other methods are needed to probe this conformational change. Site- ...

In vitro random mutagenesis, followed by phenotype screening, provides a rapid and convenient tool for identifying novel genes involved in the phenotype of interest. However, the forward mutagenic approach in mammalian somatic cells is seriously limited by the diploidic nature of the g ...

The expansion of the genetic alphabet, by an unnatural base pair system, provides a powerful tool for the site-specific incorporation of extra, functional components into nucleic acids by replication and transcription. We developed several unnatural base pairs that function in PCR and ...

Random mutagenesis is one of the most effective methodologies to generate variant libraries for directed protein evolution. Indeed, this approach requires no structural or mechanistic information and can uncover unexpected beneficial mutations. Here, we describe a new random mu ...

Oligonucleotide-mediated mutagenesis is a useful tool for engineering nucleotide changes at defined positions in a DNA sequence. Oligonucleotide-based approaches are commonly used to introduce missense mutations at individual codons in a gene or gene segment, thereby reveal ...

Bacterial artificial chromosomes are used to maintain and modify large sequences of different origins in Escherichia coli. In addition to RecA-based shuttle mutagenesis, Red recombination is commonly used for sequence modification. Since foreign sequences, such as antibiotic ...

Random mutagenesis is a useful technique to study the functions of different gene products. Propagation of the genes cloned in plasmids through a mutator strain, like Escherichia coli XL1-red, produces randomly mutagenized plasmid libraries. This method offers a very simple and econo ...

Random mutagenesis is widely used in protein engineering to improve or alter protein function. Creating random mutant libraries typically requires cloning of randomly mutagenized fragments into an expression vector, which is laborious and often hampered by lack of unique and conve ...

This chapter contains brief notes on molecular genetics, focusing on those aspects most frequently encountered in genetic epidemiology. The main sections cover the organisation and physical structure of genetic material, the mechanisms involved in transmitting genetic mate ...

Epidemiology as defined by Last is “the study of the distribution and determinants of health-related states or events in specified populations and the application of this study to the prevention and control of health problems”. Traditional epidemiological studies include quantit ...