Identification of F1 Cassava (&lt;i&gt;Manihot esculenta&lt;/i&gt; Crantz) Progeny Using Microsatellite Markers and Capillary Electrophoresis

MSI analysis is becoming increasingly important for the detection of both hereditary non-polyposis colorectal cancer and sporadic primary colorectal tumours with MSI high phenotype. The Bethesda panel of five microsatellite markers has been proposed to provide uniform criteria for MSI analysis. Here we report on an MSI analysis approach using quasimonomorphic mononucleotide repeats and denaturating high performance liquid chromatography (DHPLC). We analysed 595 newly diagnosed colorectal tumours and 145 normal samples. Microsatellite markers BAT-25, BAT-26, NR-21, NR-22, and NR-27 were amplified in multiplex reaction and analysed using DHPLC and capillary electrophoresis (CE). DHPLC conditions for analysis of MSI multiplex assay were evaluated and tested. Analysis and cross-examination of the results obtained from 96 samples using DHPLC and capillary electrophoresis showed the same sensitivity and specificity of the two approaches for detecting MSI-H tumours. Using our new approach we showed that the tested markers are quasimonomorphic in a Slovenian population, with frequencies of polymorphisms 0.07%, 1.4%, 2.1%, 1.4%, and 1.4% for BAT-25, BAT-26, NR-21, NR-22, and NR-27, respectively. Forty-three (7.2%) new MSI-H tumours were identified, of which 84% showed instability in all 5 tested markers. Overall, we developed a high-throughput, robust, accurate and cost-effective approach for the detection of MSI-H tumours.

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Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: the Tuebingen experience

Leukemia ◽

10.1038/sj.leu.2402761 ◽

2003 ◽

Vol 17 (1) ◽

pp. 237-240 ◽

Cited By ~ 67

Author(s):

H Kreyenberg ◽

W Hölle ◽

S Möhrle ◽

D Niethammer ◽

P Bader

Keyword(s):

Capillary Electrophoresis ◽

Stem Cell ◽

Quantitative Analysis ◽

Stem Cell Transplantation ◽

Microsatellite Markers ◽

Cell Transplantation ◽

Fluorescence Detection ◽

Allogeneic Stem Cell Transplantation ◽

Pcr Amplification

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Comparison of Gel Versus Capillary Electrophoresis for Chimerism Monitoring by PCR Amplification of Microsatellite Markers after Allogeneic Hematopoietic Cell Transplantation.

Blood ◽

10.1182/blood.v104.11.5108.5108 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5108-5108

Author(s):

Robert Zeiser ◽

Alexandros Spyridonidis ◽

Ralph Waesch ◽

Hartmut Bertz ◽

Jurgen Finke

Keyword(s):

Capillary Electrophoresis ◽

Microsatellite Markers ◽

Cell Transplantation ◽

Hematopoietic Cell Transplantation ◽

Hematopoietic Cell ◽

Allogeneic Hematopoietic Cell Transplantation ◽

Mixed Chimerism ◽

Unrelated Donor ◽

Preferential Amplification ◽

Limiting Dilution

Abstract Hematopoietic chimerism has been demonstrated to be relevant for donor cell engraftment and detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). In the light of increasing numbers of aHCT as a treatment modality, rapid, reliable and cost effective chimerism monitoring techniques acquire novel relevance. We evaluated the informativeness of five microsatellite markers in 376 donor/recipient pairs and compared the ability of polyacrylamide gel electrophoresis (GE) and capillary electrophoresis (CE) to detect mixed chimerism (MC) after aHCT. The sensitivity for GE and CE with respect to different markers was determined by limiting dilution assays with MC samples containing defined amounts of cells or DNA. Furthermore, CE was applied in 37 retrospectively selected patients with a MC detected by GE, having undergone aHCT for different hematological diseases and initially achieving a complete donor chimerism. The sensitivity and accuracy quantified by limiting dilution was higher CE as compared to GE with all microsatellites. Potential pitfalls with the combined application of CE was preferential amplification, leading to the misdiagnosis of CC in the presence of MC and the occurrence of stutter peaks in the representative area. In case of preferential amplification with the initially applied marker D1S80, evaluation with a second microsatellite was beneficial. Investigation of the selected pts samples demonstrated that detection of an increasing MC was earlier with CE as compared to GE. The detected recipient genotype by CE examination, despite a negative GE result, ranged from 0.7% to 7.1%. We conclude that chimerism assessment with our five microsatellites identified informative alleles in 99% of all donor/recipient pairs. CE displayed a higher sensitivity and accuracy when compared to GE and additionally allowed for detection of an increasing MC suggestive for relapse. Prospective clinical investigation with CE for the predictive value of an increasing MC for graft failure or relapse is necessary. Informativeness of the institutional microsatellite marker panel No of pairs/ markers D1S80 THO1 D14S120 YNZ22 SE33 Numbers indicate patients were the marker was informative (ratio in %). rel: related donor; unrel: unrelated donor; n.t.: not tested; non inf.: non-informative. 165 rel/211 unrel 87 (53%)/145 (69%) n.t. n.t. n.t. n.t. 78 rel/66 unrel non inf. 22 (22%)/20 (30%) n.t. n.t. n.t. 56 rel/46 unrel non inf. non inf. 18 (32%)/16 (35%) n.t. n.t. 38 rel/20unrel non inf. non inf. non inf. 17 (45%)/15 (75%) n.t. 21 rel/5 unrel non inf. non inf. non inf. non inf. 19 (90%)/4 (80%) rel vs unrel 163/165 (98%) vs 210/211 (99%)

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