One of the major challenges in microbiological research is identification of the microbe to the strain level. Often we rely on physical and biochemical characteristics for such identification. However, with the advance of biotechnological methods, newer and improved tools have been developed to suit the task.
The major ones in these DNA fingerprinting techniques are
• Pulsed Field Gel Electrophoresis (PFGE)
• Randomly Amplified Polymorphic DNA (RAPD)
• Amplified Fragment Length Polymorphism (AFLP)
• Amplified Ribosomal DNA Restriction Analysis (ARDRA)
• Repetitive extragenic palindromic PCR (Rep-PCR)
• Triplicate Arbitrary Primed PCR (TAP-PCR)
• DNA microarrays
1. Pulsed Field Gel Electrophoresis (PFGE)
RFLP analysis requires this technique to separate the genomic fragments according to their size. In RFLP, the genome is cut using restriction enzymes into few fragments of different sizes. These fragments are size-fractionated using PFGE. This method is widely used for finding specific changes such as DNA deletion, insertions etc. This has been found to be effective in finding the differences between the related strains of bacteria. RFLP is also combined with other techniques such as flow cytometry for bacterial strain identification. This is more sensitve, and can be automated easily.
This uses Southern hybridization along with r-DNA targeted probing. The probes can be either partial sequences, spacer regions or whole rDNA sequence. DNA extracted from the bacterial colonies are subjected to restriction and probed with rRNA operon sequence.
The pattern thus produced is compared with the reference library. The variations between the bacterial strains exhibit variations in the position and intensity of the rRNA bands thus formed.
3. Randomly Amplified Polymorphic DNA (RAPD)
Arbitrary primers which can bind to complementary sequences are used to find the genetic profile. If the binding is at a site where amplification of DNA segments is possible, finger prints are generated. The temperature of annealing, primer choice, purity of the DNA sequence etc determine the precision and accuracy of detecting variations within the genome.
4. Amplified Fragment Length Polymorphism (AFLP)
This technique combines RFLP and PCR based methods. Primer recognitions (adapter) sequences are added to the pool of DNA fragments. The genomic DNA is digested using two different restrictions enzymes. One is a frequent cutter with a higher cutting frequency so that large number of fragments can be produced. The other enzyme is a rare cutter. These fragments are bound to the primer recognitions sequences. The primer sequences are specific to the adapter sequences and serve to specifically amplify those sequences to yield unique amplification patterns of genome of microbes. The profiles can also be compiled and analyzed with a reference library using software tools. The results can be compared on a larger scale.
5. Amplified Ribosomal DNA Restriction Analysis (ARDRA)
This method involves 16sRDNA sequences. These are amplified and restriction analysis with five enzymes is done. The elecrophoretic pattern reveals the genomic profile which is compared with the profile in the genomic library.
6. Repetitive extragenic palindromic PCR (Rep-PCR)
This is suitable for rapid identification of microorganisms.The technique employs primers specific to the repetitive DNA elements in prokaryote and eukaryote genomes. These primers are allowed to anneal to the specific complementary sequences of the genome and they are selectively allowed to amplify using the PCR. The products are then fractionated using the agarose gel electrophoresis and profiles of the microbes are created. This is then analyzed with a reference library of such sequences using softwares such as BioNumerics.
Repeated cluster analysis and genetic algorithms add to the efficiency of the process. The temperature of annealing and elongation can also be sufficiently adjusted to improve the performance.
7. Triplicate Arbitrary Primed PCR (TAP-PCR)
This is a recent method for profiling and producing transcriptionally active PCR products. The first step is attaching unique TAP DNA sequences to the 5'a dn 3' ends of the gene using TAP PCR primers. The second step is a nested PCR which appends the promoter and terminator to the DNA fragments.
The method has been widely used in transfection assays. TAP fragments are found to have potentials for developing as DNA vaccines.
8. DNA microarrays
The bacterial DNA is isolated and allowed to anneal with an array of DNA sequences on a chip/microarray. The bound sequences are identified and analyzed based on their expression pattern and a profile is formed. The DNA probes are fixed onto a solid support. The absence / presence of a gene is determined from the fluorescent expression of hybridized DNA microarray after scanning. The procedure costs a little expensive, but is the most robust. Genome wide microarrays are now employed for microbial strain identification especially in epidemiological studies.
Effective identification however, employs a combination of two or more of the above methods and improved versions of these techniques. A comparative and collective approach has been found to provide the best results.
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