Construction of cDNA libraries with low stringency pcr and hybrid primers and cloning and a Schistosoma mansoni apirase

Author(s)

Juliana Lopes Rangel Fietto

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2001-03-14

Examining board members

Sergio Verjovski Almeida; Emmanuel Dias Neto; Hamza Fahmi Ali El Dorry; Chuck Shaker Farah; Lucile Maria Floeter Winter

• Advisor: Sérgio Verjovski Almeida

Abstract

This work shows purification of an apyrase from the tegument membrane of S. mansoni adult worms that exhibited an ADP/ATP hydrolysis ratio near 2. MALDI-TOF analyses showed that helicase and carboxylesterase were present in the sample, however in the literature there is no ADPase activity described for either enzyme. Western blot analyses showed that the purified apyrase ha no epitopes common to members of the CD39 apyrase family. On the other hand, a CD39-like apyrase remained in the insoluble fraction that was not used in the purification procedures. This work studied the construction of cDNA libraries with Hybrid Consensus-degenerate Oligonucleotide Primers (40bp) and low stringency RT-PCR. Introduction of touch-down PCR in the standard ORESTES technique improved the number of non-redundant sequences per library twofold. Association of touch-down with hybrid consensus-degenerated oligonucleotide primers increased this number to 300 sequences per library. The modifications resulted in a 10-fold decrease in the amount of work required for libraries construction and high-throughput sequencing (Fietto et al., 2000; addendum to patent application #196,716 - Ludwig Institute for Cancer Research). The best amplification profiles were obtained in RT-PCR reactions with low salt and a slow temperature ramp. Libraries made with shorter hybrid primers (25bp) showed more redundant sequences, reflecting a higher specificity for few templates. These shorter primers were used in combination with tegument mRNA to clone a gene fragment homologous to CD39 of mouse. This sequence was used in RACE-PCR reactions (rapid amplification of cDNA ends) to clone a 2.9 Kb cDNA corresponding to most of the predicted coding sequence of the Schistosoma gene. (AU)