Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats.

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.

Appendix. Cloning and sequence of the gene encoding enzyme E-1 from the methionine salvage pathway of Klebsiella oxytoca.

The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.

A frameshift mutation at the junction of an IS1 insertion within lacZ restores beta-galactosidase activity via formation of an active lacZ-IS1 fusion protein.

The insertion of IS1 elements into lacZ results in the loss of beta-galactosidase activity, and such insertions exert a severe polar effect on the expression of the distal genes of the operon. In addition to these properties, the mutation lacZ::IS1-MS319 has the unique property of reversion to Lac+ (ts) spontaneously or after treatment with the frameshift mutagen ICR-191; such revertants retain the IS1 element. We have determined that the site of integration of IS1 into lacZ is at position 4338, 18 nucleotides from the end of the sequence encoding the C-terminus of beta-galactosidase. Reversion to Lac+ promoted by ICR-191 results from the loss of a G residue from a GGG sequence located at the junction of lacZ and IS1. As a result an active, but temperature-sensitive, lacZ-IS1 fusion protein is formed containing six amino acids derived from IS1 which replace six amino acids encoded by lacZ. The IS1 element in MS319 is a new member of the iso-IS1 family, which we designate IS1T.