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Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations.

Kalogianni DP, Bravou V, Christopoulos TK, Ioannou PC, Zoumbos NC - Nucleic Acids Res. (2007)

Bottom Line: Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line.We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively.A single K562 cell was detectable amidst 10(6) normal leukocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Patras, Patras 26500, Greece.

ABSTRACT
We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 10(6) normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

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Dipstick test for the detection of PML-RARa (left panel) and AML1-ETO (right panel) transcripts. The target DNA was diluted in PCR buffer (67 mM Tris-HCl, pH 8.8, 16.6 mM (NH4)2SO4 and 0.1 ml/l Tween-20). The results obtained by agarose (2%) gel electrophoresis and ethidium bromide staining are also presented for direct comparison. N = RT negative (water instead of RNA in the RT mixture); P1 = patient negative for the PML-RARa translocation; P2 = patient positive for the PML-RARa translocation; P3 and P4 = patients positive for the AML1-ETO translocation. NB4 = RNA from NB4 cell line, that carries the PML-RARa translocation.
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Figure 5: Dipstick test for the detection of PML-RARa (left panel) and AML1-ETO (right panel) transcripts. The target DNA was diluted in PCR buffer (67 mM Tris-HCl, pH 8.8, 16.6 mM (NH4)2SO4 and 0.1 ml/l Tween-20). The results obtained by agarose (2%) gel electrophoresis and ethidium bromide staining are also presented for direct comparison. N = RT negative (water instead of RNA in the RT mixture); P1 = patient negative for the PML-RARa translocation; P2 = patient positive for the PML-RARa translocation; P3 and P4 = patients positive for the AML1-ETO translocation. NB4 = RNA from NB4 cell line, that carries the PML-RARa translocation.

Mentions: The detection of the translocation PML-RARa was based on the analysis of the cell line NB4, one patient positive for the translocation and a patient negative for the translocation. The results are shown in Figure 5. A faint red band appears in the test zone of the strip at 0.4 fmol of amplified DNA (40 pM) and the intensity increases with the amount of loaded DNA, up to 400 fmol. The corresponding electropherogram shows a faint zone at 6.4 fmol of amplified DNA.Figure 5.


Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations.

Kalogianni DP, Bravou V, Christopoulos TK, Ioannou PC, Zoumbos NC - Nucleic Acids Res. (2007)

Dipstick test for the detection of PML-RARa (left panel) and AML1-ETO (right panel) transcripts. The target DNA was diluted in PCR buffer (67 mM Tris-HCl, pH 8.8, 16.6 mM (NH4)2SO4 and 0.1 ml/l Tween-20). The results obtained by agarose (2%) gel electrophoresis and ethidium bromide staining are also presented for direct comparison. N = RT negative (water instead of RNA in the RT mixture); P1 = patient negative for the PML-RARa translocation; P2 = patient positive for the PML-RARa translocation; P3 and P4 = patients positive for the AML1-ETO translocation. NB4 = RNA from NB4 cell line, that carries the PML-RARa translocation.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851627&req=5

Figure 5: Dipstick test for the detection of PML-RARa (left panel) and AML1-ETO (right panel) transcripts. The target DNA was diluted in PCR buffer (67 mM Tris-HCl, pH 8.8, 16.6 mM (NH4)2SO4 and 0.1 ml/l Tween-20). The results obtained by agarose (2%) gel electrophoresis and ethidium bromide staining are also presented for direct comparison. N = RT negative (water instead of RNA in the RT mixture); P1 = patient negative for the PML-RARa translocation; P2 = patient positive for the PML-RARa translocation; P3 and P4 = patients positive for the AML1-ETO translocation. NB4 = RNA from NB4 cell line, that carries the PML-RARa translocation.
Mentions: The detection of the translocation PML-RARa was based on the analysis of the cell line NB4, one patient positive for the translocation and a patient negative for the translocation. The results are shown in Figure 5. A faint red band appears in the test zone of the strip at 0.4 fmol of amplified DNA (40 pM) and the intensity increases with the amount of loaded DNA, up to 400 fmol. The corresponding electropherogram shows a faint zone at 6.4 fmol of amplified DNA.Figure 5.

Bottom Line: Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line.We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively.A single K562 cell was detectable amidst 10(6) normal leukocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Patras, Patras 26500, Greece.

ABSTRACT
We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 10(6) normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

Show MeSH
Related in: MedlinePlus