Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells.
The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).
Kumar
Gyanendra
G
GE Healthcare Life Sciences, Piscataway, NJ 08854, USA. gyan.kumar@ge.com
Garnova
Elena
E
Reagin
Michael
M
Vidali
Andrea
A
D017103:Bacillus Phages / Q000201:enzymology*; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats; D018598:Minisatellite Repeats; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011933:Reagent Kits, Diagnostic
D011933:Reagent Kits, Diagnostic