Sources of fetal samples for prenatal diagnosis

The aim of PND is to provide a result as early in the pregnancy as possible, and thus it is preferable if the fetal sample is obtained within the first trimester by CVS sampling between 10-12 weeks of pregnancy. The alternative is to obtain an amniocentesis sample from about 14-16 weeks of pregnancy. Overall CVS samples provide more DNA and are less prone to co-sampling of maternal cells, which can lead to contamination of the fetal DNA.

The yield of DNA extracted from a trophoblast sample weighing around 20-25 mg is typically 30-40 ^g. DNA can be prepared from amniotic fluid cells directly or after culturing. The quantity of DNA extracted directly from 15 ml of non-cultivated amniotic fluid cells (~5 ^g) is sufficient for PND based on PCR methods in most cases.

The risk of contamination with maternal DNA can be ruled out in most cases by the presence of one maternal and one paternal allele following the amplification of 'informative' polymorphic repeat markers, as mentioned above in the best practice guidelines.

The analysis of single-cells within the context of PGD involves a different procedure in that the single-cell is simply lysed in the eppendorf tube in which the cell was initially placed. This releases the cellular DNA content and the reagents for the initial processing are added directly to the tube; these are usually for the first-round PCR reaction, although some protocols start with whole genome amplification or multiple displacement amplification. A protocol for lysing single cell samples is outlined within the protocol described for single-cell genotype analysis (see 'Cell Lysis' in 17.5).

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