SIMULTANEOUS DNA-FINGERPRINTING, DIAGNOSIS OF SEX AND SINGLE-GENE DEFECT STATUS FROM SINGLE CELLS

Sex and cystic fibrosis status have been previously diagnosed separate ly at the single cell level. We have developed a sensitive, reliable, accurate and rapid (within 5-6 h) system for the simultaneous diagnosi s of sex, cystic fibrosis and a DNA 'fingerprint' within a single reac tion from a variety of single cells. As contamination cannot be totall y excluded, particularly at the single cell level, DNA 'fingerprinting ' can be used to assess the risk of contamination. High sensitivity wi th single cells is combined with very high specificity (estimated matc hing probability of 10(-7)-10(-8)), allowing the source of the amplifi ed cell to be identified with a very high degree of probability. Fluor escent primers were multiplexed for six tetranucleotide microsatellite sequences to determine the DNA fingerprint; the amelogenin gene was u sed to diagnose sex, and primers for the CFTR region were used to dete rmine cystic fibrosis (CF) status. Analysis of the fluorescent product was undertaken using an automated DNA sequencer with Genescan softwar e, This technique has many applications such as prenatal and preimplan tation diagnosis, forensic identification of small or degraded samples , and detection of contamination sources. DNA fingerprints of single h aploid spermatozoa and other cells can be assessed, so ensuring the de tection of both diploid and haploid contamination during preimplantati on diagnosis.