Techniques of molecular biology
DNA animation
For more extensive list on protein methods, see protein methods. For more extensive list on nucleic acid methods, see nucleic acid methods.
Molecular cloning
Main article: Molecular cloning
Transduction image
Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector. This recombinant DNA technology was first developed in the 1960s. In this technique, a DNA sequence coding for a protein of interest is cloned using polymerase chain reaction (PCR), and/or restriction enzymes, into a plasmid (expression vector). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker (usually antibiotic resistance). Additionally, upstream of the MCS are the promoter regions and the transcription start site, which regulate the expression of cloned gene. # ISO certification in India
This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection, electroporation, microinjection and liposome transfection. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection.# ISO certification in India
DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.# ISO certification in India
Polymerase chain reaction
Main article: Polymerase chain reaction
Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific DNA sequence to be copied or modified in predetermined ways. The reaction is extremely powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. PCR has many applications, including the study of gene expression, the detection of pathogenic microorganisms, the detection of genetic mutations, and the introduction of mutations to DNA. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-directed mutagenesis. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, quantitative PCR which allow for quantitative measurement of DNA or RNA molecules.# ISO certification in India
Two percent agarose gel in borate buffer cast in a gel tray.
Gel electrophoresis
SDS-PAGE
Main article: Gel electrophoresis
Gel electrophoresis is a technique which separates molecules by their size using an agarose or polyacrylamide gel. This technique is one of the principal tools of molecular biology. The basic principle is that DNA fragments can be separated by applying an electric current across the gel – because the DNA backbone contains negatively charged phosphate groups, the DNA will migrate through the agarose gel towards the positive end of the current. Proteins can also be separated on the basis of size using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis. # ISO certification in India
Proteins stained on a PAGE gel using Coomassie blue dye.
The Bradford Assay
Main article: The Bradford Assay
The Bradford Assay is a molecular biology technique which enables the fast, accurate quantitation of protein molecules utilizing the unique properties of a dye called Coomassie Brilliant Blue G-250. Coomassie Blue undergoes a visible color shift from reddish-brown to bright blue upon binding to protein. In its unstable, cationic state, Coomassie Blue has a background wavelength of 465 nm and gives off a reddish-brown color. When Coomassie Blue binds to protein in an acidic solution, the background wavelength shifts to 595 nm and the dye gives off a bright blue color. Proteins in the assay bind Coomassie blue in about 2 minutes, and the protein-dye complex is stable for about an hour, although it’s recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation. The concentration of protein in the Bradford assay can then be measured using a visible light spectrophotometer, and therefore does not require extensive equipment. # ISO certification in India
This method was developed in 1975 by Marion M. Bradford, and has enabled significantly faster, more accurate protein quantitation compared to previous methods: the Lowry procedure and the biuret assay. Unlike the previous methods, the Bradford assay is not susceptible to interference by several non-protein molecules, including ethanol, sodium chloride, and magnesium chloride. However, it is susceptible to influence by strong alkaline buffering agents, such as sodium dodecyl sulfate (SDS). # ISO certification in India