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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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Study of the reproducibility of the dipstick test using amplification products from the BCR-ABL translocation (two PCR pools) and total RNA from K562 cells.
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Figure 8: Study of the reproducibility of the dipstick test using amplification products from the BCR-ABL translocation (two PCR pools) and total RNA from K562 cells.

Mentions: The reproducibility of the strip assay was assessed by assaying 6.4 and 50 fmoltarget DNA (PCR products) five times (Figure 8). The strips were then scanned (desktop scanner ScanJet 4300C, Hewlett Packard) and the intensity of the test zone was estimated densitometrically. The coefficient of variation (CV) (relative standard deviation) of the intensity was 7.6 and 4.9%, respectively. To determine the overall reproducibility of the test, three samples containing 12.8 pg, 1.6 × 103 pg and 106 pg RNA from Ph1 cells were subjected, in quadruplicate, to reverse transcription, nested PCR and strip assay (Figure 8). The test zones of the strips were analyzed by scanning and densitometry as above and the CV were found to be 29, 10.7 and 8.5%, respectively. Finally, we assessed the reproducibility of the detection of 1 K562 cell in the presence of 106 normal cells. The CV after scanning and densitometry of the test zones of the strips was 22% (n = 4).Figure 8.


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)

Study of the reproducibility of the dipstick test using amplification products from the BCR-ABL translocation (two PCR pools) and total RNA from K562 cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 8: Study of the reproducibility of the dipstick test using amplification products from the BCR-ABL translocation (two PCR pools) and total RNA from K562 cells.
Mentions: The reproducibility of the strip assay was assessed by assaying 6.4 and 50 fmoltarget DNA (PCR products) five times (Figure 8). The strips were then scanned (desktop scanner ScanJet 4300C, Hewlett Packard) and the intensity of the test zone was estimated densitometrically. The coefficient of variation (CV) (relative standard deviation) of the intensity was 7.6 and 4.9%, respectively. To determine the overall reproducibility of the test, three samples containing 12.8 pg, 1.6 × 103 pg and 106 pg RNA from Ph1 cells were subjected, in quadruplicate, to reverse transcription, nested PCR and strip assay (Figure 8). The test zones of the strips were analyzed by scanning and densitometry as above and the CV were found to be 29, 10.7 and 8.5%, respectively. Finally, we assessed the reproducibility of the detection of 1 K562 cell in the presence of 106 normal cells. The CV after scanning and densitometry of the test zones of the strips was 22% (n = 4).Figure 8.

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