遗传学实验技术

As described in a different chapter in this volume, the uracil-specific excision reaction (USER) fusion method can be used to assemble multiple small DNA fragments (∼0.75-kb size) into larger 3-kb DNA segments both in vitro and in vivo (in Escherichia coli). However, in order to assemble an entire sy ...

Many bacterial and archaeal genomes are of a similar size to molecules that have been cloned in the yeast Saccharomyces cerevisiae and thus might be clonable as single, circular episomes in this host. Yeast offers a variety of efficient tools for the manipulation and study of cloned DNA. One strate ...

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology, and synthetic biology. We developed an error-correcting recursive construction procedure that attempts to address this challe ...

We describe here a method for sequence- and ligation-independent cloning (SLIC). SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. These fragments are then assembled in vitro and transformed into Escherichia coli to g ...

Synthetic biologists have adopted the engineering principle of standardization of parts and assembly in the construction of a variety of genetic circuits that program living cells to perform useful tasks. In this chapter, we describe the BioBrick standard as a widely used method. We prese ...

Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific e ...

Overlap extension or fusion PCR is thought to be a simple and easy method to produce fusion DNA fragments without the need for restriction enzyme digestion and DNA ligation. However, this method has not been used frequently, probably as it is not always reliable. When natural sequences are used for ov ...

Ability to manipulate the genome or design genes with desired mutation is critical for functional studies. Recombineering has made genetic manipulation of large genomic fragments very feasible and efficient. In the bacteriophage lambda-based recombineering system, three pro ...

The promise of synthetic biology lies in the creation of novel function from the proper combination of genetic elements. De novo gene synthesis has become a cost-effective method for building virtually any conceptualized genetic construct, removing the constraints of extant sequen ...

This chapter presents TmPrime, a computer program to design oligonucleotide for both ligase chain reaction (LCR)- and polymerase chain reaction (PCR)-based de novo gene synthesis. The program divides a long input DNA sequence based on user-specified melting temperatures and assemb ...

The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than where they have been identified originally. Gene synthesis often provides a fast and economically efficient approac ...

The International Genetically Engineered Machines (iGEM) competition allows undergraduate teams to develop projects in synthetic biology within the context of a large, international Jamboree. Organizing and managing a successful iGEM team is an exercise in advanced agile pro ...

Build-a-Genome is an intensive laboratory course at Johns Hopkins University that introduces undergraduates to the burgeoning field of synthetic biology. In addition to lectures that provide a comprehensive overview of the field, the course contains a unique laboratory component ...

It is generally assumed that genetic engineering advances will, inevitably, facilitate the misapplication of biotechnology toward the production of biological weapons. Unexpectedly, however, some of these very advances in the areas of DNA synthesis and sequencing may enable the i ...

Genomic data analysis in evolutionary biology is becoming so computationally intensive that analysis of multiple hypotheses and scenarios takes too long on a single desktop computer. In this chapter, we discuss techniques for scaling computations through parallelization of cal ...

Open-source software (OSS) encourages computer programmers to reuse software components written by others. In evolutionary bioinformatics, OSS comes in a broad range of programming languages, including C/C++, Perl, Python, Ruby, Java, and R. To avoid writing the same functionality mul ...

The emergence of genomics tools for the evolutionary and comparative biology community led to a rapid explosion in the number of online resources targeted at this specialized community, including Web-based comparative genomics software, such as the Artemis Comparison Tool (WebACT ...

Genetical genomics combines acquired high-throughput genomic data with genetic analysis. In this chapter, we discuss the application of genetical genomics for evolutionary studies, where new high-throughput molecular technologies are combined with mapping quantitati ...

This chapter describes bioinformatic tools for analyzing epigenome differences between species and in diseased versus normal cells. We illustrate the interplay of several Web-based tools in a case study of CpG island evolution between human and mouse. Starting from a list of orthologo ...

Metagenomics is the study of microbial organisms using sequencing applied directly to environmental samples. Similarly, in metatranscriptomics and metaproteomics, the RNA and protein sequences of such samples are studied. The analysis of these kinds of data often starts by asking t ...