Nucleic Acid Blotting
Nucleic acid blotting is a well-established technique for locating a genomic region, gene, or other sequence of interest from a complex mixture of DNA or RNA. The first blotting method was named after its inventor, the British biologist Edwin Southern.
Southern blotting is used to detect a sequence in a DNA mixture, and Northern blotting detects a sequence in an RNA mixture. Southern and Northern blotting are the two most common nucleic acid blotting techniques.
- Southern blotting can be used in mapping genes and has applications such as detecting large gene rearrangements/deletions and large trinucleotide repeat expansions.
- Northern blotting enables the measurement of gene expression, for example in research studies regarding differentiation, morphogenesis, and genetic abnormalities.
Blotting involves electrophoretic separation of nucleic acids in a gel, transfer of the separated components to a membrane, and detection of specific sequences by probing the membrane. The transfer and immobilization on the membrane make the nucleic acids available to probes used for detection, which do not readily enter the gel.
There are other nucleic acid blotting methods, such as dot blots, slot blots, and colony/plaque lifts, that do not include electrophoresis. Dot and slot blots involve direct application of the sample to the membrane, which is then probed. Colony and plaque lifts involve direct transfer of colonies of microorganisms from a culture plate onto a membrane. The microorganisms are then lysed in situ and probed. These techniques are quick and may give sufficient information when detection is not complicated by interfering molecules, or demands an initial separation step.
Alternative techniques such as the Polymerase Chain Reaction (PCR) have superseded blotting in many applications that require the sensitive detection or quantitation of nucleic acid sequences in complex mixtures. Despite this, Southern blotting still has its advantages, for example in determining the position of sequences in large fragments, detecting chromosomal rearrangements, picking up related sequences with lower homology, and determining the number of gene copies.
Northern blotting has a low sensitivity compared to PCR-based techniques, but a high specificity, thus reducing the risk of false positives. Northern blotting also enables the detection of novel splice variants, whereas PCR-based investigations of splicing events may require prior knowledge of putative splice sites.
Blotting consists of many steps. To ensure success, work backwards in a systematic manner - make sure that the detection step works on raw material before running gels and transferring nucleic acids to the membrane.
In Southern blotting, accurate location of sequences requires careful preparation of intact genomic DNA, or well-controlled fragmentation using efficient restriction enzymes. Separation, transfer, and detection may require some optimization since the process is sensitive to fragment size.
In Northern blotting, great care should be taken to avoid degradation of RNA by ubiquitous RNases.