Multiple displacement amplification on single cell and possible PGD applications.
Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.
King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. hellani@kfshrc.edu.sa
Ali
A
Hellani
Serdar
S
Coskun
Moncef
M
Benkhalifa
Abelghani
A
Tbakhi
Nadia
N
Sakati
Ali
A
Al-Odaib
Pinar
P
Ozand
D000483:Alleles; D004247:DNA / Q000032:analysis; D004314:Down Syndrome / Q000175:diagnosis; D005260:Female; D005914:Globins / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D018598:Minisatellite Repeats / Q000235:genetics; D009154:Mutation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D019836:Preimplantation Diagnosis; D011296:Prenatal Diagnosis / Q000379:methods*; D014314:Trisomy / Q000175:diagnosis
D005914:Globins; D004247:DNA