![]() ![]() ![]() Investigating the absolute error for pairs of 116 H37Rv lanes ( Figure 2), we found that the error was consistently higher in gel-to-gel comparisons than in comparisons of lanes from the same gel. Means and two standard errors of the mean error bars for pairwise comparisons among 116 12-banded H37Rv lanes show that error is consistently larger when comparing lanes between gels than when. ![]() The resulting collections of fragment lengths for each lane (bacterial isolate or laboratory strain H37Rv) were exported to our ACM software and compared with other lanes.įigure 2. The Whole Band Analyzer software quantitated fragment lengths for the IS 6110-visualized bands, which were inspected and edited manually in the software package. The resulting images were aligned with three registration marks that gave reference to the original nylon membrane. Two films' exposures were scanned into Whole Band Analyzer software (BioImage, Ann Arbor, MI, USA) one was obtained when probing for the internal standard, the other when probing for IS 6110. Internal standards were used to quantitate fragment sizes visualized with a DNA probe to IS 6110. Computer-assisted comparison and clustering were performed on the RFLP lanes as described in Woelffer et al., 1996 (9). tuberculosis isolates with IS 6100-based RFLP fingerprinting was performed as described in van Embden et al., 1993 (8). We also explored improving the specificity among matched fingerprints to reveal additional information in RFLP lanes. We developed an approach to identifying a set of identical fingerprints when the identity of the fingerprints is nontransitive. ![]() The parameters of the computational ACM are adjusted to provide the same high sensitivity as the labor-intensive visual inspection used over the last 5 years. The computational approach to lane comparison-align-and-count method (ACM)-permits calculation of the number of fragments that match between two IS 6110-based RFLP fingerprints. We developed an automated computational system, in which a statistical analysis of the error in measuring fragment sizes provides a conceptual framework for comparing sets of fragment lengths. Studies in Ethiopia, Tunisia, and The Netherlands (1), South Africa (2), India (3), Denmark and Greenland ( 4, 5), the United States (6), and Tanzania (7), among many others, exploit IS 6110-based RFLP genetic fingerprints. Such molecular epidemiologic studies provide information about tuberculosis (TB) transmission patterns. 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. The combination of conventional epidemiologic investigations with molecular techniques for genotyping pathogens has elucidated the epidemiology of many infectious diseases. ![]()
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