Scanning for nucleotide variations in mitochondrial DNA fragments of Schistosoma japonicum by single-strand conformation polymorphism

Abstract

In this study, we employed a mutation scanning approach for the direct visual display of genetic variability in mitochondrial DNA (mtDNA) fragments within and among populations of Schistosoma japonicum from the People's Republic of China. Fragments of the NADH dehydrogenase 1 gene (ND1) and the cytochrome c oxidase subunit I (COI) were individually amplified from parasite DNA by polymerase chain reaction (PCR), denatured and subjected to single-strand conformation polymorphism (SSCP) analysis. Using ND1 and COI fragments, individuals representing different genotypes could be readily identified based on characteristic and reproducible SSCP profiles. The results demonstrated the usefulness of SSCP for the direct visual display of low-level sequence variation in mtDNA of S. japonicum prior to DNA sequence analysis. This approach has important implications for studying the genetic structure and biology of S. japonicum populations, and for analysing the inheritance of mitochondrial DNA.

Dideoxy fingerprinting of low-level nucleotide variation in mitochondrial DNA of the human blood fluke,Schistosoma japonicum

“…The enhanced display of sequence variation in ddF compared with, for example, SSCP and sequencing is largely the result of additional mobility shifts (i.e., SSCP components) and changes in band intensities following the dideoxy component(s) [8], and results in a higher mutation detection rate. For instance, a previous study (see [17]) demonstrated that SSCP was not always able to reliably differentiate samples (e.g., Sj4M1 and Sj4F10) differing by a point mutation in the ND1, whereas in this study, ddF allowed the sample Sj4M1 to be distinguished from SjF10 by the loss of a band at position 348 and a mobility shift as a consequence of the loss of a termination segment (see Fig. 1).…”

“…As expected, single nucelotide differences located toward the 3¢-end of the ND1 fragment (e.g., at nucleotide position 426; cf. [17]) were not detected as a consequence of the 5-terminal location of the primer binding site and the length of the fragment (404 bp). Nonetheless, if the detection or location of all nucleotide variations over the entire length of the ND1 fragment were required, then the current protocol could be readily adapted to incorporate 1±2 (internal) termination reaction primers, or by using bidirectional ddF [18].…”

“…Although all amplicons were of the same size, they were known to differ in sequence by 1±6 nucleotides [17]. In ddF, variable samples could be readily differentiated from one another by their characteristic profiles (Fig.…”

“…A 404 bp fragment of the ND1 was amplified by polymerase chain reaction (PCR) [16] from 5±10 ng genomic DNA with primers rgND1F (forward: 5¢-CGATGGTTA-TACTGTTCATAGT-3¢) and rgND1R (reverse: 5¢-CACCA-TAATCAAATGGAGAACG-3¢) [17]. PCR reactions (50 mL) were performed in 10 mM Tris-HCl, pH 8.…”

“…The ND1 amplicons were purified over spin columns (Wizard TM PCR Preps; Promega) and then subjected to a cycle sequencing reaction (¦mol TM kit; Promega) using primer rgND1F and ddATP, the latter being chosen because of the relatively uniform spacing of termination fragments in the ND1 sequence [17]. Products were then mixed with equal volumes of loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanole), denatured at 94 o C for 5 min, snapcooled on a freeze block (±20 o C) for 2 min, and then 3 mL were subjected to electrophoresis (7 W, 22 o C, 19 h) in prerun (30 min) 0.4 mm thick, 0.6´mutation detection enhancement (MDE; FMC BioProducts, Rockland, ME, USA) gels.…”