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.
Hellani
Ali
A
King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. hellani@kfshrc.edu.sa
Coskun
Serdar
S
Benkhalifa
Moncef
M
Tbakhi
Abelghani
A
Sakati
Nadia
N
Al-Odaib
Ali
A
Ozand
Pinar
P
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