Tandem Repeat Polymerase Chain Reaction Research Articles

Extraction and amplification of nuclear and mitochondrial DNA from ancient and artificially aged bones

An experimental design is presented that simulates an accelerated process of DNA degradation in human bone tissues and thus provides a possibility for a systematic investigation of factors hampering DNA extraction and amplification. Equal sized slices of human femoral bones were incubated in 90 °C water for 2 h up to 30 days. DNA was extracted and subjected to a human specific Duplex polymerase chain reaction (PCR) and also to a Multiplex short tandem repeat (STR) PCR. Additionally 24 ancient bones representing different age periods were investigated in the same way. The results were compared to those from the artificially aged samples. After just 12 h of incubation, DNA is totally degraded, but still fully typable. After 36 h no reproducible amplification of DNA is possible. Using Multiplex PCR the DNA from artificially aged bones shows the typical STR pattern for ancient samples suggesting that the in vitro approach provides a useful and comparable method to elucidate the DNA degradation process in bones.

GM-CSF receptor targeted treatment of primary AML in SCID mice using Diphtheria toxin fused to huGM-CSF.

The severe combined immunodeficient (SCID) mouse model may be used to evaluate new approaches for the treatment of acute myeloid leukemia (AML). We have previously demonstrated the killing of SCID mouse leukemia initiating cells by in vitro incubation with human GM-CSF fused to Diphtheria toxin (DT-huGM-CSF). In this report, we show that in vivo treatment with DT-huGM-CSF eliminates AML growth in SCID mice. Seven cases of AML were studied. SCID mice were treated intraperitoneally with the maximally tolerated dose of 75 microg/kg/day for 7 days. Antileukemic efficacy was determined at days 40 and 80 after transplantation, by enumerating the percentages of human cells in SCID bone marrow using flow cytometry and short tandem repeat polymerase chain reaction (STR-PCR) analysis. Four out of seven AML cases were sensitive to in vivo treatment with DT-huGM-CSF at both evaluation time points. In three of these cases, elimination of human cells was demonstrated by flow cytometry and STR-PCR. One AML case showed moderate sensitivity for DT-huGM-CSF, and growth of the two remaining AML cases was not influenced by DT-huGM-CSF. Sensitivity was correlated with GM-CSFR expression. Our data show that DT-huGM-CSF can be used in vivo to reduce growth of AML and warrant further development of DT-huGM-CSF for the treatment of human AML.