While formaldehyde reacts with free amines of the bases producing a Schiff base that is unable to hydrogen bond to complementary baes preventing the formation of secondary structures. Glyoxal reacts with guanine and introduces an additional ring that prevents base-pairing by steric hindrance. After extraction for Northern blotting, already single-stranded RNA needs to be pretreated with formaldehyde or a denaturing solution with glyoxal to prevent the formation of base-paired secondary structures. Given the instability of RNA, procedures need to be done with sterile, RNAse-free supplies that usually require reagents to be autoclaved or filter-sterilized for autoclavable plastics – 20 mins at 120☌ to inactivate RNAses, for non-autoclavable plastics – 2 or more hours in 0.1% diethyl pyrocarbonate, glassware needs to be heated at 180☌ for 3 h. However, RNA detection by Northern blot requires different considerations and a pretreatment step because the end-user is using RNA which is naturally unstable. Northern blots omit the need for restriction digest. If DNA is clonally derived, a digestion time of 1-2h may be sufficient. DNA samples are digested with appropriate restriction enzyme for 2-24h at 37☌. Southern blotting requires an additional step because of the need to take purified DNA and partially digest it into smaller, different sized, double-stranded fragments with restriction enzymes (endonucleases) that cut the DNA at specific spots. Purified whole genomic DNA, plasmid DNA or RNA transcripts are isolated using common extraction techniques such as organic extractions (phenol-chloroform and ethanol precipitation), filter-based, spin basket extractions (glass fiber, derivitized silica or ion exchange column-based method) and magnetic particle methods.Īfter extraction, Southern and Northern blot protocols begin to differ. Both blotting methods share a similar workflow 1) Sample preparation of purified high quality DNA or RNA from a sample, 2) Gel electrophoresis to separate nucleic acid fragments by size, 3) transfer (“blotting”) to a solid-support that immobilizes isolated target nucleic acid, 4) preparation & hybridization of nucleic acid probe, and lastly 5) the detection of the nucleic acid probe.ĭuring sample preparation, Southern and Northern blots both begin very similarly - the target DNA or RNA is purified from samples through nucleic acid extraction. Shortly after southern blotting was developed, the premise of this analytical technique was applied to the measurement of the size and amount of RNA transcripts from a gene of interest which is known as Northern Blotting. It is an analytical technique in molecular biology research that end-users use to measure the size and amount of specific DNA sequences in a complex mixture through immobilization of the target sequence to a solid-support followed by hybridization of a complementary DNA probe. Southern blotting was originally introduced by Edwin Southern in 1975. Below, we review some considerations between Southern, Northern and Western blots used to detect DNA, RNA or protein, respectively.ĭetection of Nucleic Acids by Southern and Northern Blotting This hybridized complex (“immobilized target molecule-label/probe”) which consists of a bound probe can be later detected and visualized using various imaging methods. End-users are then able to incorporate “labels” (radiolabel, fluorescent label, reporter enzyme label) to the immobilized targets with probes that are sequence-specific or shape-specific to the target molecule of interest (either DNA, RNA or protein) during an incubation step that facilitates hybridization. Then, these separated molecules are transferred to a solid membrane (nitrocellulose, nylon, polyvinylidene difluoride (PVDF), etc.) suited to immobilize the target molecule of interest. Initially, an electrophoretic procedure is used to separate molecules (or protein and nucleic acid fragments) by size on a gel based on the movement of macromolecules in an electric field. All blotting techniques share a similar workflow. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules. Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules.
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