Simultaneous DNA‘fingerprinting’, diagnosis of sex and single-gene defect status from single cells

Molecular Diagnostics in Preimplantation Genetic Diagnosis

“…One such strategy is the use of linked markers 46,60,61 which simultaneously controls for contamination. 62 Use of one or two linked markers reduces undetected ADO by approximately 50% and 75% respectively and with three linked markers ADO is virtually always detected. 46 The use of linked markers carries considerable advantages not only from the point of view of reducing the possibility of misdiagnosis, but also by potentially increasing the number of embryos available for transfer.…”

“…Compared with nested PCR, FPCR combines increased sensitivity and throughput, shorter turnaround time, 73 and superior precision in fragment sizing. 62 The use of a laser system to perform automated fragment analysis with various fluorescent molecules, each with their own unique wavelength of emitted light, allows simultaneous discrimination of unrelated, similarly sized products. Furthermore, the thousandfold increase in sensitivity 74 compared with ethidium bromide allows a single round of PCR, potentially avoiding the contamination which can result from multiple tube openings.…”

“…Several different instruments are available for such analyses and the technique has been successfully applied to PGD development and clinical cases in many laboratories. 55,62,73 Fluorescent PCR is also compatible with many established forms of mutation analysis such as SSCP, 76 ARMS, 77 and restriction enzyme digestion. 78 …”

“…82 It may also be possible to assess similar numbers of loci in single cells but to date the maximum number of sequences amplified simultaneously from a single cell is seven using either conventional ethidium detection 12 or fluorescent PCR. 62 Unfortunately the problems of allele dropout and preferential amplification persist even with the FPCR approach. 40,80 Multiplex PCR can also be used to detect ADO by simultaneous amplification of a disease causing mutation and an informative "linked" polymorphism.…”

Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosis

“…When analysed by PCR this involves the successful amplification of a specific DNA copy of each chromosome. Although the efficiency and the accuracy of the diagnosis procedure has increased with the introduction of fluorescent PCR 3,4 and multiplex PCR, 5,6 a low risk of misdiagnosis persists. 7 Another difficulty is the development of multiplex PCR from a single cell.…”