6^th Molecular Biology Webinar

Techniques of Molecular Biology

Molecular cloning

One of the most primary techniques of molecular biology to study protein feature is molecular cloning. On this approach, DNA coding for a protein of interest is cloned the usage of polymerase chain reaction (PCR), and/or restriction enzymes right into a plasmid (expression vector). A vector has three distinctive functions: a beginning of replication, a multiple cloning site (MCS), and a selective marker usually antibiotic resistance. Positioned upstream of the more than one cloning site are the promoter areas and the transcription start site which adjust the expression of cloned gene. This plasmid may be inserted into both bacterial and animal cells. Introducing DNA into bacterial cells may be executed by means of transformation via uptake of bare DNA, conjugation through cell-cell contact or by way of transduction through viral vector. Introducing DNA into eukaryotic cells, along with animal cells, by bodily or chemical manner is called transfection. Several exceptional transfection techniques are available, consisting of calcium phosphate transfection, electroporation, microinjection and liposome transfection. The plasmid can be included into the genome, ensuing in a stable transfection, or may stay unbiased of the genome, referred to as transient transfection.

Polymerase chain reaction

Polymerase chain reaction (PCR) is a very versatile technique for copying DNA. In brief, PCR permits a specific DNA sequence to be copied or modified in predetermined ways. The reaction is extremely effective and under ideal conditions could expand one DNA molecule to grow to be 1.07 billion molecules in much less than two hours. The PCR technique may be used to introduce limit enzyme sites to ends of DNA molecules, or to mutate precise bases of DNA, the latter is a way called site-directed mutagenesis. PCR can also be used to determine whether a selected DNA fragment is determined in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, quantitative PCR which permit for quantitative dimension of DNA or RNA molecules.

Macromolecule blotting and probing

The terms northern, western and eastern blotting are derived from what to begin with became a molecular biology joke that played on the term Southern blotting, after the approach defined with the aid of Edwin Southern for the hybridization of blotted DNA. Patricia Thomas, developer of the RNA blot which then has become called the northern blot, actually did not use the term.