Simultaneous DNA 'fingerprinting', diagnosis of sex and single-gene defect status from single cells.
Sex and cystic fibrosis status have been previously diagnosed separately at the single cell level. We have developed a sensitive, reliable, accurate and rapid (within 5-6 h) system for the simultaneous diagnosis of sex, cystic fibrosis and a DNA 'fingerprint' within a single reaction from a variety of single cells. As contamination cannot be totally excluded, particularly at the single cell level, DNA 'fingerprinting' can be used to assess the risk of contamination. High sensitivity with single cells is combined with very high specificity (estimated matching probability of 10(-7)-10(-8)), allowing the source of the amplified cell to be identified with a very high degree of probability. Fluorescent primers were multiplexed for six tetranucleotide microsatellite sequences to determine the DNA fingerprint; the amelogenin gene was used to diagnose sex, and primers for the CFTR region were used to determine cystic fibrosis (CF) status. Analysis of the fluorescent product was undertaken using an automated DNA sequencer with Genescan software. This technique has many applications such as prenatal and preimplantation diagnosis, forensic identification of small or degraded samples, and detection of contamination sources. DNA fingerprints of single haploid spermatozoa and other cells can be assessed, so ensuring the detection of both diploid and haploid contamination during preimplantation diagnosis.
Findlay
I
I
Institute of Epidemiology and Health Services Research, University of Leeds, UK.
Urquhart
A
A
Quirke
P
P
Sullivan
K
K
Rutherford
A J
AJ
Lilford
R J
RJ
D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics; D016172:DNA Fingerprinting; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods*; D013094:Spermatozoa / Q000473:pathology