scholarly journals Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences

2009 ◽  
Vol 75 (14) ◽  
pp. 4747-4752 ◽  
Author(s):  
Pablo Alvarado ◽  
Jose L. Manjón
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Fragment Size ◽  
Polymorphism Analysis ◽  
Internally Transcribed Spacer ◽  
Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.

scholarly journals Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

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