Computer-simulated virtual RFLP patterns generated from in silico digestions of phytoplasma 16S rRNA genes from strains detected in this study belonging to groups: (A) 16SrI using HhaI
(MH428961: 16SrI-P *); (B) 16SrIII using BstUI (MH428959: 16SrIII-F *); (C) 16SrIX using RsaI (MH428962:16SrIX-A[infinito]); (D) 16SrXIII using MseI (MH428958: 16SrXIII-H *); (E, F) 16SrXV using HaeIII and HpalI (MH428963: 16SrXV-A[infinito]), obtained using iPhyClassifier tool.
For RFLP, the URA5 gene amplified product was subjected to double enzymatic digestion using the Sau96I (5000 U/mL) and HhaI
(20000 U/mL) enzymes (New England BioLabs, USA) incubated at 37[degrees]C in dry for 3 hours.
The 54Thr polymorphism abolishes the HhaI
enzyme restriction site resulting in un-cut 180bp band.
Los cebadores amplifican un fragmento de 557 pb del gen gmk y despues de la digestion con la enzima HhaI
, se generan fragmentos de 429 y 124 pb para el ST5 mientras que para el ST8 se generan fragmentos de 323, 124, y 105 pb.
Restriction endonuclease analysis using HhaI
and HpaII to discriminate among group B Pasteurella multocida associated with haemorrhagic septicaemia.
Kod genotipa e2/e2 javljaju se fragmenti od 91 bp i 83 bp jer HhaI
ne sece GTGC region cisteinskih rezidua.
Restriction endonuclease analysis for characterization of serotypes of hemorrhagic septicaemia can be done with the enzyme HhaI
. Discrimination of the isolates can be done by application of ribotyping as well as large DNA separation by means of pulsed-field gel electrophoresis.
Following the supplier's instructions, aliquots of 5-|mL amplified PCR products were digested with 2 U HhaI
restriction enzyme (MBI Fermentas, Germany) at 37[degrees]C for 10 h.
Two (mu) L of each amplicon were digested with EcoRI, AluI, HhaI
and TaqI endonucleases, according to the manufacturers" instruction (Promega, USA) at 37degC (65degC for TaqI) overnight.