The number of IS 6110 fingerprints continues to increase, with many studies across the globe producing IS 6110 data to characterize Mycobacterium tuberculosis isolates. We present an approach by which identical patterns can be identified from large collections of DNA fingerprints. These limitations significantly constrain the size, scope, and standardization of molecular epidemiologic investigations. Furthermore, the results of computer-assisted matching are not as reproducible as systematic computational methods. This approach is accurate but cumbersome and excessively labor-intensive as the number of isolates exceeds several hundred. For larger numbers, commercially available computer programs can be used to identify a manageable subset of potentially matching patterns, which are then compared visually. Fewer than 100 patterns can be compared visually. The most frequently used genotyping techniques (e.g., pulsed-field gel electrophoresis, restriction fragment length polymorphism, and randomly amplified polymorphic DNA) yield fragment-based data. Therefore, more samples can be analyzed in a shorter time.Īn alternative name for the technique is Cleaved Amplified Polymorphic Sequence (CAPS) assay.The combination of conventional epidemiologic investigations with molecular techniques for genotyping pathogens has elucidated the epidemiology of many infectious diseases. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive. In species with low polymorphism rates, additional restriction endonucleases can be tested to increase the chance of finding polymorphism. Typically, in species with moderate to high polymorphism rates, two to four restriction endonucleases are used such as EcoRI The probes are screened for RFLPs using genomic DNA of different genotypes digested with restriction endonucleases.Southern blots of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences.Digests of the plasmids are screened to check for inserts.The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for example, pUC18).Total DNA is digested with a methylation-sensitive enzyme (for example, PstI), thereby enriching the library for single- or low-copy expressed sequences ( PstI clones are based on the suggestion that expressed genes are not methylated).INDELs can create or abolish restriction endonuclease (RE) recognition sites, thus affecting quantities and length of DNA fragments resulting from RE digestion. Genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.). Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes. Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.Īn RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. Restriction Fragment Length Polymorphism (RFLP) Introduction
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