Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells

Abstract

We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1-110 mutation leading to beta thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF-PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri-allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF-PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.

Rapid Prenatal Diagnosis by QF‐PCR: Evaluation of 30,000 Consecutive Clinical Samples and Future Applications

“…28 It is also possible to include primers to detect point mutations causing single gene defects at the same time as testing aneuploidies. 17 Recently, in a preliminary study, we have successfully investigated the diagnostic value of using a CGH microarray for the detection of chromosome abnormalities by comparing its results with those obtained by QF-PCR and conventional cytogenetics. 29 At present, many new noninvasive approaches are also under investigations, including the detection of fetal DNA or RNA in maternal blood.…”

“…rochromes in order to be amplified and analyzed in the same multiplex QF-PCR reactions. 17 In the course of this study the two centers developed different combinations of primers in multiplex QF-PCR reactions in order to simplify sample handling and data analysis. 10,11,13,18 Fetal sex and chromosomes X, Y copy numbers were assessed for all cases by amplification of the homologous gene AMXY together with the pseudoautosomal X22 and chromosome X-specific markers (TABLE 1).…”

Rapid prenatal diagnosis of common chromosome aneuploidies by QF‐PCR, results of 9 years of clinical experience

“…The quantitative fluorescent polymerase chain reaction (QF-PCR) assay has been developed in the last 15 years to detect major numerical chromosome disorders in a few hours after the collection of the samples (Mansfield, 1993;Pertl et al, 1994Pertl et al, , 1996Pertl et al, , 1997Pertl et al, , 1999aAdinolfi et al, 1995Adinolfi et al, , 1997Sherlock et al, 1998;Verma et al, 1998;Cirigliano et al, 1999Cirigliano et al, , 2001bCirigliano et al, , 2004Adinolfi and Sherlock, 2001a).…”

Preimplantation genetic diagnosis for myotonic dystrophy type 1: detection of crossover between the gene and the linked marker APOC2

“…A fully informative couple should have no alleles in common at the polymorphic site. The informative polymorphism allows the calculation of all possible zygote genotypes and any deviation from these combinations would be indicative of contamination (Sherlock et al, 1998;Findlay et al, 1995). If the STR marker is linked to the disease locus, and DNA is available from affected or unaffected relatives then the phase can be determined (i.e.…”