An alternative strategy to generate coding sequence of macrophage migration inhibitory factor-2 of Wuchereria bancrofti.
OBJECTIVE
Different developmental stages of Wuchereria bancrofti, the major causal organism of lymphatic filariasis (LF), are difficult to obtain. Beside this limitation, to obtain complete coding sequence (CDS) of a gene one has to isolate mRNA and perform subsequent cDNA synthesis which is laborious and not successful at times. In this study, an alternative strategy employing polymerase chain reaction (PCR) was optimized and validated, to generate CDS of Macrophage migration Inhibitory Factor-2 (wbMIF-2), a gene expressed in the transition stage between L3 to L4.


METHODS
The genomic DNA of W. bancrofti microfilariae was extracted and used to amplify the full length wbMIF-2 gene (4.275 kb). This amplified product was used as a template for amplifying the exons separately, using the overlapping primers, which were then assembled through another round of PCR.


RESULTS
A simple strategy was developed based on PCR, which is used routinely in molecular biology laboratories. The amplified CDS of 363 bp of wbMIF-2 generated using genomic DNA splicing technique was devoid of any intronic sequence.


CONCLUSIONS
The cDNA of wbMIF-2 gene was successfully amplified from genomic DNA of microfilarial stage of W. bancrofti thus circumventing the use of inaccessible L3-L4 transitional stage of this parasite. This strategy is useful for generating CDS of genes from parasites that have restricted availability.
Nikhil
N
Chauhan
Division of Microbiology & Immunology, Vector Control Research Centre (ICMR), Puducherry; Scientist 'G' and Director-in-Charge, Regional Medical Research Centre (ICMR), Nehru Nagar, Belagavi 590 010, Karnataka, India.
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SL
Hoti
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004605:Elephantiasis, Filarial / Q000175:diagnosis* / Q000235:genetics* / Q000469:parasitology; D005091:Exons / Q000235:genetics; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D014958:Wuchereria bancrofti / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity
D018076:DNA, Complementary; D008263:Macrophage Migration-Inhibitory Factors