The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice.

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.

Long-term RNase P-mediated inhibition of HIV-1 replication and pathogenesis.

Advances in genetic analysis and a greater understanding of human immunodeficiency virus (HIV) molecular pathogenesis have identified critical viral targets for gene interference strategies. RNase P molecules have been proposed as a novel approach for gene targeting based upon their potent catalytic activity, as well as versatile external guide sequence (EGS) which can be modified to specifically recognize almost any target mRNA. We designed a truncated EGS to specifically recognize the highly conserved U5 region of HIV-1 mRNA and mediate subsequent cleavage of hybridized mRNA by the RNase P enzyme component. The active U5-EGS (560), as well as a disabled U5 EGS (560D) control, were cloned into plasmids containing proviral constructs and transfected into a CD4(+) T cell line that was thereafter infected with HIV-1 MN. CD4(+) T cells treated with the active U5 EGS (560) were observed to maintain CD4(+) expression and did not produce HIV p24 gag antigen, form syncytia or undergo apoptosis up to 30 days after infection. Identical cells expressing the inactivated form of the U5 RNase P EGS completely down-regulated CD4 expression, produced elevated levels of HIV-1, formed large syncytia and underwent apoptosis similar to untreated cells. HIV-1 replication and related cytopathology can be effectively inhibited in CD4(+) T cells expressing a protective U5 EGS (560).

Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR.

A rapid and sensitive strategy for the specific identification of Mycobacterium tuberculosis (TB) was designed and evaluated using crude mycobacterial lysates. The speed of real-time polymerase chain reaction (PCR) was combined with the sensitivity of fluorogenic probes to confirm the presence of mycobacteria as well as specifically identify the presence of members of the mycobacteria tuberculosis complex (MTC) in a single-tube assay. Oligonucleotides were designed to amplify the internal transcribed spacer (ITS) from several mycobacterial species. Specific fluorogenic probes were included in the PCR reaction for the identification of TB as well as Mycobacterium bovia and Mycobacterium africanum in bacterial lysates. The combination of TB-specific fluorogenic probes with real-time PCR formed an approach determined to be fast (less than 40 min), sensitive (less than 800 copies of DNA) and reliable for the specific detection of the MTC. Our data demonstrate the use of real-time PCR and fluorogenic probes in a rapid and sensitive assay to distinguish members of the MTC from other mycobacterial species.

Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLA-A2.

Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.