Leland Stanford Junior University

Dartmouth College

John Charles Boothroyd

James Lawrence Burg

Philippe Jacques Pouletty

Lloyd H Kasper

Kami Kim

Dominique Soldati

Roland Buelow

L David Sibley

A method is provided for multiplying the number of copies of a target polynucleotide sequence comprising a series of primer hybridization, extending, and denaturing steps to provide an intermediate double-stranded DNA molecule containing a promoter sequence (through the use of a promoter-sequence-containing primer) incorporated upstream from the target sequence. The double-stranded DNA intermediate is then used to grow multiple RNA copies of the target sequence. The resulting RNA copies can be used as target sequences to produce further copies. Multiple cycles of this sort can thereby exponentially increase the number of target sequence copies.

Selective amplification of target polynucleotide sequences

A method of transient transfection of obligate intracellular parasites is described. This method is exploited to develop a system for stable transformation utilizing selectable genes. For example, introduction of the chloramphenicol acetyl transferase (CAT) gene fused to Toxoplasma flanking sequences followed by chloramphenicol selection results in parasites stably expressing CAT. DNA hybridization analysis indicated that the CAT gene had inserted via homologous recombination.

Transfection and genetic manipulations in obligate intracellular

Genetic material encoding p30 and B1 peptides of Toxoplasma gondii has been isolated and characterized. This genetic material allows the production of peptides for use in diagnosis or immunization or can itself be directly used in hybridization assays.

P30 peptides of Toxoplasma gondii

Diagnostic genes for toxoplasmosis

Diagnostic assays based on the discovery of dimorphism in the p30 antigen and gene and the direct association of this dimorphism with the known bimodal virulence pattern. Assays can be based on different antigenic behavior of virulent (referred to here as type I) and avirulent (type II) p30 antigens or on the differences in the corresponding genes. There is a one-to-one relationship between the type I and type II antigen/genes and virulence. Specific genetic materials, specific antibodies, and analytical techniques that allow a diagnosis to be made between virulent and avirulent infections are disclosed.

Growing community of inventors

Palo Alto, CA, United States of America

John Charles Boothroyd