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DNA extraction and amplification of leishmania from archived, Giemsa-stained slides, for the diagnosis of cutaneous Leishmaniasis by PCR.

DNA was isolated from 92 Giemsa-stained smears of lesions from suspected cases of cutaneous leishmaniasis and used for PCR-based diagnosis of Leishmania infection. Each smear had been examined under a light microscope at x 1,000 and scored for amastigote numbers. Although the smears had been stored for up to 4 years, all the microscopy-positive slides were also positive by PCR and four of the 14 smears that were negative by microscopy (although of lesions that were clinically consistent with leishmaniasis) were also PCR-positive. PCR-based investigations therefore appear to offer an effective method to confirm suspected cases of cutaneous leishmaniasis using (even archived) samples that have been collected, from humans (and reservoir hosts) in the field, by simple methods.

Animals↗

Long-term room temperature storage of high-quality embryonic stem cell genomic DNA extracted with a simple and rapid procedure.

A very simple procedure for the isolation of high-quality, high-molecular-weight genomic DNA from embryonic stem cells is described. The DNA is very stable once dried and can be stored for long periods of time without refrigeration. Living cells are lysed in a sodium dodecyl sulfate and EDTA buffer containing proteinase K and then air-dried. Samples can be processed in bulk, and an individual can easily process thousands of samples for extraction and shipment on a daily basis using only common laboratory materials such as plastic ware and a multichannel pipetteman.

Alleles↗

Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.

Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.

Autoradiography↗