17 - Y Chromosome-Specific PCR: Maternal Blood

Abstract

This chapter presents a Y chromosomal PCR amplification to correlate the presence of an amplified band of DNA with a male fetus as part of a continuing assessment of the efficacy and purity of a variety of fetal cell separation techniques. The fetal cell diagnostic work is based on a combination of fetal cell enrichment by multi-parameter flow sorting, followed by PCR for sequences present on the Y chromosome. This approach is sensitive enough to detect one fetal cell, and the signal-to-noise ratio can be controlled by the duration of film exposure. It also allows the extent of background false positive amplification to be determined. Venous blood (20 mL) is collected in citrate dextrose (ACDA) from a pregnant woman, which is diluted 1:3 with Hanks' balanced salt solution (HBSS), layered over a FicolI-Hypaque column, and centrifuged at 2000 rpm for 20 min at room temperature in a Sorvall RT 6000 centrifuge. Fetal cell analysis and sorting are performed on a Becton-Dickinson FACS Star Plus with a Lysis II software program. The gain is standardized manually using fluorescent beads and a fluorescence-conjugated antibody to an antigen not expressed on human cells, keyhole limpet hemocyanin.