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.

The high incidence of STR D21S1446 homozygosity in Han and She populations living in South Eastern China

“…The presence of disomic and diallelic STRs is important for accurately determining STR copy numbers. A normal chromosomal number would result in a PCR pattern in which the ratio of the alleles is approximately 1:1 [7,19]. Trisomy would be indicated by the presence of a tri-allelic pattern with a ratio of 1:1:1 or trisomic diallelic pattern of 2:1 [7,19,20].…”

“…A normal chromosomal number would result in a PCR pattern in which the ratio of the alleles is approximately 1:1 [7,19]. Trisomy would be indicated by the presence of a tri-allelic pattern with a ratio of 1:1:1 or trisomic diallelic pattern of 2:1 [7,19,20]. A high degree of homozygosity at a given STR, for example D21S1446, would result in an increased chance of a 2:1 diallelic pattern, possibly resulting in false positives for trisomy.…”

Assessment of new markers for the rapid detection of aneuploidies by quantitative fluorescent PCR (QF–PCR)

“…The procedures for the QF-PCR amplification and testing of the samples were those previously described (Sherlock et al 1998 ;Cirigliano et al 1999). Briefly PCR amplifications were performed in a final volume of 25 µl containing 5 µl of the extracted DNA, 200 µmol dNTPs, from 4 to 35 pmol of each primer, 1 Unit Taq (Promega) in 2 m MgCl # .…”

Multiplex PCR: Optimization and Application in Diagnostic Virology

“…Multiplex PCR has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility. Since its introduction, multiplex PCR has been successfully applied in many areas of nucleic acid diagnostics, including gene deletion analysis (19,20), mutation and polymorphism analysis (86,96), quantitative analysis (94,124), and RNA detection (51,126). In the field of infectious diseases, the technique has been shown to be a valuable method for identification of viruses, bacteria, fungi, and/or parasites.…”