Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involved a mutation pre-screening step to limit the ...

Reverse transcription-PCR (RT-PCR) describes a technique whereby RNA is first reverse transcribed into complementary DNA (cDNA) using the enzyme reverse transcriptase, and the resulting cDNA amplified in either a single-step or a two-step nested PCR reaction. It is particularly use ...

Making use of the nucleotides sequence of the RNA genome (7,440 nt) of poliovirus, synthetic deoxyoligonucleotides, 60–70 nt in length are synthesized. The oligonucleotides that map to adjacent segments in the genome are designed such that they are of plus- and minus-strand polarity with the o ...

The manual design of synthetic genes is a tedious and error-prone process—even for very short genes—and it becomes completely infeasible when multiple synthetic genes are needed. GeneDesign is a set of modules that automate batch nucleotide manipulation. Here, we explain the installat ...

The yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides can overlap by as few as 20 bp and can be as long as 200 nucleotides in length to prod ...

Recombineering is a recombination-based highly efficient method of genetic engineering. It can be used to manipulate the bacterial chromosomal DNA as well as any episomal DNA. Recombineering can be used to insert selectable or nonselectable DNA fragments and subclone DNA fragments w ...

De novo gene synthesis allows the creation of custom DNA molecules without the typical constraints of traditional cloning assembly: scars, restriction site incompatibility, and the quest to find all the desired parts to name a few. Moreover, with the help of computer-assisted design, the pe ...

This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer ...

The throughput of DNA reading (i.e., sequencing) has dramatically increased recently owing to the incorporation of in vitro clonal amplification. The throughput of DNA writing (i.e., synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To over ...

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 ...