Derivation of the complete msp4 gene sequence of Anaplasma marginale without cloning

Abstract

The gene encoding MSP4, a 31-kDa surface protein of the rickettsia Anaplasma marginale, was completely sequenced without prior cloning of the gene. Degenerate oligodeoxyribonucleotides (oligos) corresponding to the N-terminal amino-acid sequence of MSP4 were used as primers in the polymerase chain reaction (PCR) to amplify a 65-bp fragment of which the central 32 bp was nondegenerate. The 32-bp probe was hybridized to restriction enzyme-digested, genomic A. marginale DNA in Southern blots, and the hybridizing fragment was size selected from agarose gels and ligated into the Bluescript II SK (+/-) plasmid. The ligation reaction mixture was used as a template for PCR, amplified from the 32-bp oligo or its inverse complement to plasmid sequences. The PCR products were sequenced to obtain the entire msp4 gene and surrounding regions. This method of sequencing a gene without previously obtaining a clone for that gene could be beneficial in situations where conventional cloning and screening strategies are inefficient or ineffective.