PCR amplification and genetic analysis in a microwell cell culturing chip
2009 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, 3465-3471 p.Article in journal (Refereed) Published
We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity
Place, publisher, year, edition, pages
2009. 3465-3471 p.
adherent cell; article; biotinylation; controlled study; culture medium; fluorescence microscopy; gene mutation; genetic heterogeneity; genetic screening; genotype; human; human cell; lab on a chip; magnetic field; magnetism; mutation rate; polymerase chain reaction; priority journal; sequence analysis; tumor suppressor gene; wild type
IdentifiersURN: urn:nbn:se:kth:diva-11663DOI: 10.1039/b912596eISI: 000272142200001PubMedID: 20024024ScopusID: 2-s2.0-72849112298OAI: oai:DiVA.org:kth-11663DiVA: diva2:279102
QC 201007282009-12-012009-12-012012-01-18Bibliographically approved