Table 3.
Precautions against Contamination in Single Cell PCR
| Type of contamination | Precautions |
|---|---|
| Operator contamination (can be cellular or product contamination; see below) | Elimination or reduction Protective clothing: gloves (close fitting), cap, overshoes, gown Frequent changes of gloves Operator technique Detection DNA Fingerprinting (incorporate informative polymorphic markers) |
| Product contamination (PCR products from previous reactions also known as “carry-over” contamination) | Elimination or reduction Dedicated equipment (PCR machine, pipettes, tubes) Dedicated reagents (all solutions) Filtration of reagents Filtered pipette tips Positive displacement pipettes Aliquot all reagents One-time use of tips and reagent aliquots UV irradiation of preparation area/equipment/reagents Autoclaving equipment and reagents Restriction enzyme digestion of PCR master mix (component reagents) Switch from nested PCR to FPCR Geographical separation of pre-PCR/PCR and post-PCR activities Preparation of PCR reagents in laminar flow Decontamination of surfaces/equipment with 10% bleach Post-PCR sterilization (dUTP and UDG/isopsoralen) Purchase reagents as ready-made ‘molecular biology grade’ solutions Reduce number of tube-opening events Mineral oil overlay Detection Switch from nested PCR to FPCR Use of multiple negative controls (cell wash blanks and reagent blanks) Test all component reagents before clinical case |
| Genomic DNA (gDNA) contamination (eg, DNA used for positive controls/assay development) | Elimination or reduction Isolate procedures involving gDNA (eg, no gDNA in reagent prep room) |
| Cellular contamination (eg, Maternal cells (cumulus), paternal cells (sperm), embryonic material (from different embryos) or operator cells (epithelial)) | Elimination or reduction Rinse embryo thoroughly (to remove cumulus cells) Exclusive use of Intracytoplasmic sperm injection for fertilization (to prevent supernumerary sperm exposure) Wash blastomere thoroughly Change micropipettes if a cell lyses during biopsy or dish-to-tube transfer Detection DNA “fingerprinting” (incorporate informative polymorphic markers) Wash blank controls |