Small-Scale Isolation of High Molecular Weight DNA from Leishmania braziliensis

Abstract

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.

Molecular and immunological characterization of L14 ribosomal protein from Leishmania braziliensis

“…Sequence analysis of randomly selected clone revealed the existence of a 286 bp long ORF showing significant homology with a region of the gene coding for L14 ribosomal proteins of different trypanosomatids. This fragment was used as a probe to screen an L. braziliensis genomic library constructed in lambda phages, as described by Carmelo et al (2000). Sequencing of a positive clone revealed the presence of the full-length L14 coding region.…”

“…Southern blot, Northern blot and PFGE analysis L. braziliensis genomic DNA was isolated by standard methods (Carmelo et al 2000). Two mg of L. braziliensis genomic DNA was fully digested with Msp I and Sph I enzymes (Roche Diagnostics, Mannheim, Germany), which digest respectively twice and once inside the predicted ORF; then with Dde I restriction enzyme (Roche Diagnostics), which cuts once, 67 bp downstream of the stop codon at the non-coding 3k end, before being size-resolved on 0 .…”