Cloned cDNA Probes

The increasing accumulation of DNA sequences in nucleic acid databases coupled with the availability of custom synthesis of oligonucleotides has provided a relatively straightforward means to design and produce gene probes and primers for PCR. Such probes and primers are usually designed with bioinformatics software, using sequence information from nucleic acid databases. Alternatively, gene-family-related sequences may also be successfully employed. However, there are many gene probes that have traditionally been derived from cDNA or from genomic sequences and that have been cloned into plasmid and phage vectors. These require manipulation before they can be labelled and used in hybridisation experiments. Gene probes may vary in length from 100 bp to a number of kilobases, although this is dependent on their origin. Many are short enough to be cloned into plasmid vectors and are useful in that they may be manipulated easily and are relatively stable, both in transit and in the laboratory. The DNA sequences representing the gene probe are usually excised from the cloning vector by digestion with restriction enzymes and purified. In this way, vector sequences that may hybridise non-specifically and cause high background signals in hybridisation experiments are removed.

RNA Gene Probes

 It is also possible to prepare cRNA probes or riboprobes by in vitro transcription of gene probes cloned into a suitable vector. A good example of such a vector is the phagemid pBluescript SK; at each end of the multiple cloning site where the cloned DNA fragment resides are promoters for T3 or T7 RNA polymerase. The vector can be linearised with a restriction enzyme digest, and T3 or T7 RNA polymerase is used to transcribe the cloned DNA fragment. Provided a labelled NTP is added in the reaction, a riboprobe labelled to a high specific activity will be produced ( Figure1). One advantage of riboprobes is that they are single-stranded and their sensitivity is generally regarded as superior to cloned double-stranded probes. They are used extensively in in situ hybridisation and for identifying and ana lysing mRNA.

Fig1. Production of cRNA (riboprobes) using T3 RNA polymerase and phagemid vectors.

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مواضيع ذات صلة

Analysing Genes and Gene Expression

Screening Gene Libraries

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity

Interphase Polytene Chromosomes Arise by DNA Amplification

Cloning Vectors : Cosmid-Based Vectors

Cloning Vectors : Phagemid-Based Vectors

Cloning Vectors : Virus-Based Vectors

Cloning Vectors : Plasmids

The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA

Chromosome Painting and DNA Sequencing Reveal the Evolution of Chromosomes