There have been a number of major advances in molecular biology in the past few years and the aim of this review is to describe some of these advances, focusing on their benefits and limitations when applied to investigating pulmonary disorders. It is written with the practising pulmonary researcher in mind, not as an introduction for the uninitiated. Useful web addresses and a list of references are included to enable interested readers to examine each technique in detail.

New technologies either improve existing techniques or develop new approaches to old questions in order to generate information more quickly, easily, accurately or in a more easily repeatable fashion then existing methods.¹ Some of the most powerful new technologies include polymerase chain reaction (PCR) advances, “difference analysis” (that is, the discovery of different gene expression patterns between different cells), transgenic/gene knockout technology, and gene delivery to tissues/gene therapy.

Advances in PCR technology

Since its introduction in the 1980s PCR has become a standard tool in biomedical research. The equipment (a thermal cycler) and reagents (thermostable polymerases, oligonucleotides, etc) required for PCR are widely available and relatively inexpensive. One advantage of PCR is its extreme sensitivity which makes possible the detection and analysis of low abundance DNAs. This is especially helpful when limited amounts of starting material are available, or when few copies of the target sequence are present. Applications of PCR include the cloning of known and novel genomic DNA and cDNA sequences, DNA sequencing, construction of mutant or chimeric DNAs, and quantification of mRNA and DNA. PCR is also used in certain methods of difference analysis

PCR BASED CLONING AND SEQUENCING METHODS

PCR has been used to facilitate cloning of known DNAs and to allow for the identification of novel DNAs. It can be performed directly on genomic DNA and on cDNA produced from mRNA (reverse transcription PCR (RT-PCR)). This eliminates the requirement for the production and screening of DNA libraries. When the target DNA sequence is known, the target DNA can easily be amplified using oligonucleotide primers based on the sequence. When only a portion of a target cDNA sequence is known, PCR can be used to amplify unknown sequences at the 5′ or 3′ ends of the known region (rapid amplification of cDNA ends (RACE) .Novel homologues of known proteins can also be identified using a method known as homology PCR^. This method relies upon the use of degenerate mixtures of oligonucleotide primers designed to recognize conserved motifs. Homology PCR can be used to clone novel members of protein families or to clone protein homologues in other species.

PCR based techniques such as allele specific PCR and PCR restriction length polymorphism (PCR-RFLP) can be used to speed the detection of genetic polymorphisms in large populations. PCR fragments can also be sequenced directly, enabling screening of populations for novel mutations. PCR has been incorporated directly into DNA sequencing technologies, making possible the sequencing of small (nanogram) amounts of target DNA and sequencing of unpurified DNA—for example, for direct sequencing of DNA from bacterial colonies or phage plaques

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Journal of Molecular Biology and Biotechnology
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