The relative proportions of fetal and total circulatory DNA eluted from the individual agarose gel sections were determined by use of a well-established TagMan[R] real-time PCR assay for the SRY gene
on the Y chromosome and the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (6).
To study the fragment-length distribution of plasma DNA, we developed two panels of quantitative PCR with a range of amplicon sizes targeting the leptin and SRY genes
. The leptin gene is located on chromosome 7 and is present in all human cells, whereas the SRY gene
is located on the Y chromosome and is present only in male cells.
The SRY gene
on the Y chromosome was used as a marker for male fetuses, and the concentration of the fetal SRY sequence was measured by a real-time quantitative PCR assay and an ABI PRISM 7700 Sequence Detector (Applied Biosystems) as described by Ohashi et al.
The median concentration of the SRY gene
in maternal plasma was 74 genome-equivalents/mL (interquartile range, 53-141 genome-equivalents/mL).
Because of insufficient material, however, only 73 samples were available, and in 1 sample, quantification of the SRY gene
failed because of an erroneous laboratory procedure.
We used 10 [micro]L of each of the 32 eluates for real-time PCR amplification of the SRY gene
Because the SRY gene
is found in all nucleated cells of males only, whereas the [beta]-globin gene is present in all nucleated cells of both males and females (6), we calculated the percentage of male DNA in a particular plasma or buffy coat sample, denoted as Y%, using the following equation:
The SRY gene
in male fetuses was used as a molecular marker for fetal DNA in maternal serum.
In separate experiments, however, LAMP was performed successfully without heat denaturation for template DNAs, such as k DNA, pBluescript II, and M13 mp18 vector DNA, and human genomic DNA (SRY gene
on chromosome Y), including commercially available material (data not shown).
Finally, using the male-specific SRY gene
as a marker for male fetuses, we successfully determined the gender of all fetuses tested.
Plasma samples from these two groups were assayed for circulating fetal DNA, using the SRY gene
on the Y chromosome as a marker, as described previously (7).
For detection of the transfer of nucleated cells and plasma DNA from the fetus to the mother, the SRY gene
that is present on the Y-chromosome of a male fetus was used as a fetal marker (22).