Some of the most important techniques used in molecular biology are as follows:
Molecular Biology techniques include characterization, isolation and manipulation of the molecular components of cells and organisms.
These components include DNA, the repository of genetic information; RNA, functional and structural part of the translational apparatus and proteins, the major structural and enzymatic type of molecule in cells.
(i) Expression cloning:
One of the most basic techniques of molecular biology to study protein function is expression cloning.
In this technique, DNA coding for a protein of interest is cloned (using PGR and/or restriction enzymes) into a plasmid (known as an expression vector).
This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.
The polymerase chain reaction is an extremely versatile technique for copying DNA. PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. PGR has many variations, like reverse transcription PGR (RT-PGR) for amplification of RNA, and, more recently, real-time PGR (QPGR) which allow for quantitative measurement of j DNA or RNA molecules.
(iii) Gel electrophoresis:
Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. Proteins can be separated on the basis of size by using SDS-PAGE (polyacrylamide ) gel.
(iv) Macromolecule blotting and probing Southern Blots:
The Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. These tools are widely used in forensic laboratories to identify individuals who have left blood or other DNA-containing material at the scene of crimes. The number of bands that hybridize to a short probe gives an estimate of the number of closely related genes in an organism.
The Northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. The RNAs on the blot can be detected by hybridizing them to a labeled probe. The intensities of the band reveal the relative amounts of specific RNA in each sample.
Immunoblots (Western Blots):
Proteins can be detected and quantified in complex mixtures using immunoblots (or Western blots). Proteins are electrophoresed, then blotted on a membrane and the proteins on the blot are probed with specific antibodies that can be detected with labeled secondary antibodies or protein.
(v) DNA microarray:
A DNA array is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragment. Arrays make it possible to put down a large quantity of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence (similar to Southern blotting).
A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling).
(vi) Antiquated technologies:
In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis (agarose or polyacrylainide), the size of DNA molecules was typically determined by rate sedimentation in sucrose gradients, a slow and labour-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used.