Application of optical trapping for cells grown on plates: optimization of PCR and fidelity of DNA sequencing of p53 gene from a single cell.
BACKGROUND
Optical trapping has traditionally been used to visually select and isolate nonadherent cells grown in suspension because cells grown in monolayers will rapidly reattach to surfaces if suspended in solution. We explored methods to slow cell reattachment that are also compatible with high-fidelity PCR.


METHODS
Using HeLa cells grown on plates and suspended after trypsinization, we measured the efficiency of capture by retention and movement of the cell by the laser. Success for removing a captured cell by pipette was determined by PCR amplification of the 5S rRNA gene. After optimizing PCR amplification of a 2049-bp region of the p53 gene, we determined PCR fidelity by DNA sequencing.


RESULTS
Addition of bovine serum albumin to suspended cells slowed reattachment from seconds to minutes and allowed efficient trapping. The success rate of removing a cell from the trap by pipette to a PCR tube was 91.5%. The 5S PCR assay also revealed that DNA and RNA that copurify with polymerases could give false-positive results. Sequence analysis of four clones derived from a single cell showed only three polymerase errors in 7200 bp of sequence read and revealed difficulties in reading the correct number in a run of 16 A:T. Comparison of the HeLa and wild-type human sequences revealed several previously unreported base differences and an (A:T)(n) length polymorphism in p53 introns.


CONCLUSIONS
These results represent the first use of optical trapping on adherent cells and demonstrate the high accuracy of DNA sequencing that can be achieved from a single cell.
Gale
James M
JM
Department of Cell Biology and Physiology, Steve Schiff Center for Skin Cancer University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. jgale@salud.unm.edu
Romero
Christopher P
CP
Tafoya
Gregory B
GB
Conia
Jérôme
J
D002469:Cell Separation; D003470:Culture Media; D004252:DNA Mutational Analysis / Q000379:methods; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012710:Serum Albumin, Bovine; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D003470:Culture Media; D016159:Tumor Suppressor Protein p53; D012710:Serum Albumin, Bovine