Plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.[1] They are double stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms .
Plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, the archetypes of which are the miniprep and the maxiprep/bulkprep.The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.
In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.
DNA extraction is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic steps in a DNA extraction:
Breaking the cells open to expose the DNA within, such as by grinding or sonicating the sample.
Removing membrane lipids by adding a detergent.
Precipitating the DNA with an alcohol — usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet on centrifugation. This step also removes alcohol-soluble salt.
Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+. This stops dnase enzymes from degrading the DNA.
Cellular and histone proteins bound to the DNA can be removed prior to its precipitation either by adding a protease or having prior to precipitation, precipitating with sodium or ammonium acetate, or extracting with a phenol-chloroform mixture.
If desired, the DNA can be resolubilized in a slightly alkaline buffer.
Detecting DNA
A diphenylamine (DPA) indicators will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95oC) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.
DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.
Using the Southern blot technique this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison and identification.
RNA interference (also called "RNA-mediated interference", abbreviated RNAi) is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to have evolved as a form of innate immunity against viruses and also plays a major role in regulating development and genome maintenance.
The RNAi pathway is initiated by the enzyme dicer, which cleaves double-stranded RNA (dsRNA) to short double-stranded fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and base-pairs with complementary sequences. The most well-studied outcome of this recognition event is a form of post-transcriptional gene silencing. This occurs when the guide strand base pairs with a messenger RNA (mRNA) molecule and induces degradation of the mRNA by argonaute, the catalytic component of the RISC complex. The short RNA fragments are known as small interfering RNA (siRNA) which are perfectly complementary to the gene to which they are suppressing as they are derived from long dsRNA of that same gene or MicroRNA (miRNA) which are derived from the intragenic regions or an intron and are thus only partially complementary. The RNAi pathway has been particularly well-studied in certain model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the flowering plant Arabidopsis thaliana.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms; synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
Historically, RNA interference was known by other names, including post transcriptional gene silencing, transgene silencing, and quelling. Only after these apparently-unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. RNAi has also been confused with antisense suppression of gene expression, which does not act catalytically to degrade mRNA but instead involves single-stranded RNA fragments physically binding to mRNA and blocking translation. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans, which they published in 199
