遗传学实验技术

Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide deta ...

Within the past 15 years, the utilisation of microarray technology for the detection of specific pathogen strains has increased rapidly. Presently, it is possible to simply purchase a pre-manufactured “off the shelf ” oligonucleotide microarray bearing a wide variety of known signatu ...

Standard polymerase chain reaction (PCR) protocols amplify relatively small fragments precluding the use of this approach when examining gross rearrangements of DNA. By using combinations of DNA polymerases, which feature either good polymerase activity or error-correction ...

Multiplex Ligation-dependent Probe Amplification (MLPA�) is a high-throughput method developed to determine the copy number of up to 50 genomic DNA sequences in a single multiplex PCR-based reaction. MLPA is easy to perform, requires only 20 ng of sample DNA and can distinguish sequences di ...

High-throughput sequencing and hybridization technologies promise new insights into the natural diversity and dynamics of microorganisms. Among these new technologies are phylogenetic oligonucleotide microarrays (phylochips) that depend on the standard molecules ...

Fluorescent cycle sequencing of PCR products is a multistage process and several methodologies are available to perform each stage. This chapter will describe the more commonly utilised dye-terminator cycle sequencing approach using BigDye� terminator chemistry (Applied Bi ...

The discovery of cell-free fetal DNA in the maternal plasma of pregnant women has facilitated the development of non-invasive prenatal diagnosis (NIPD). This has been successfully implemented in diagnostic laboratories for Rhesus typing and fetal sex determination for X-linked di ...

Advances in high-throughput sequencing techniques had presented a significant challenge to the processing capabilities of genetic laboratories. However, recent developments in the field of semi-automated mutation detection have revolutionised the task of mutation dete ...

QF-PCR refers to the amplification of chromosome-specific polymorphic microsatellite markers using fluorescence-labelled primers, followed by semi-quantitative analysis of the products on a genetic analyser to determine copy number and/or imbalances of specific chrom ...

This chapter outlines the methodology for the detection of mesenchymal-tumour-specific translocations in formalin-fixed paraffin-embedded tissue (FFPET) using the reverse transcriptase-polymerase chain reaction (RT-PCR). It includes the design of appropriate pri ...

Venous thrombosis affects one in one thousand people each year, and in many countries, it is a major cause of morbidity and death in hospitalised patients. Factor V Leiden and the prothrombin c.20210GA transition are relatively common in the Western World, and both increase the risk of venous thro ...

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 ...