Various strains of E. coli competent cells are widely used for gene cloning and expression. The choice of cells in which to express proteins is important; they need to allow high-levels of stable recombinant proteins to be produced that are non-toxic to the host. Where proteins are toxic they need to be tightly controlled and induced to express the protein only when required.
There are many methods for the analysis of DNA, the most sensitive however, is with the use of PCR, as it allows the amplification of a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. This can be semi-quantitative (end point PCR) or quantitative (real-time PCR), with applications in a wide variety of areas including genotyping, cloning, mutation detection, sequencing, microarrays and forensics.
Polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is often referred to as end point PCR as measurements are made once the PCR has finished (e.g. by electrophoresis in an agarose gel).
Proteomics is the large-scale experimental analysis of proteins. It is more complicated than genomics because an organism's genome is more or less constant, whereas the proteome differs from cell to cell and from time to time. Cell expression is looked at via mRNA analysis, but this does not always correlate with protein content, as mRNA is not always translated into protein and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present.
Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR), is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. Real-time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green) or sequence specific probes (i.e. Molecular Beacons or TaqMan® Probes).
RNA analysis frequently involves the detection of RNA transcripts. In order to detect RNA transcripts, the RNA is initially amplified by reverse transcription to produce complementary DNA (cDNA). The cDNA is then used to carry out numerous applications including traditional PCR amplification, real-time PCR and DNA microarray experiments. Bioline’s range of RNA Analysis products are manufactured and packaged under the most stringent conditions and are guaranteed to be RNase/DNase free.
Genotyping is the process of determining differences in the genetic make-up of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools such as sequencing or PCR and real-time PCR for high throughput.