Optimized Real-Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma

Abstract

Background: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods. Methods: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene. Results: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%-22% when amplifying DYS14 compared with 26%-140% for SRY. Conclusions: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting singlecopy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.

Noninvasive Diagnosis of Fetal Gender: Utility of CombiningDYS14andSRY

“…Although noninvasive gender determination has been reported to be an accurate and useful test in clinical practice (Devaney et al, 2011), its accuracy varies according to the protocols used with sensitivity and specificity ranging from 31% to 100% ( Johnson et al, 2004). Different Y chromosome targets, such as DYZ3 (Honda et al, 2001), AMELXY (Zhu et al, 2005), SRY (Birch et al, 2005), DYS14 (Zimmermann et al, 2005) and DAZ (Fernández-Martínez et al, 2012) have been used for sexing on cff DNA from maternal plasma. Multiple studies have achieved sensitivities in excess of 90% for gender determination with SRY (Avent and Chitty, 2006) and DYS14 (Zimmermann et al, 2005;Avent and Chitty, 2006).…”

Development and Validation of Multiplex Real-Time PCR Assay for Noninvasive Prenatal Assessment of Fetal RhD Status and Fetal Sex in Maternal Plasma

“…The DYS14 multicopy sequence on the Y chromosome, adapted from Zimmermann et al [20], was also included in the multiplex rt-PCR assay. TaqMan MGB probes were labeled with different fluorochromes allowing all sequences to be analyzed in a single multiplex reaction (table 1).…”

Increased Y-Chromosome Detection by SRY Duplexing

“…The DYS14 assay has an approximate nine-fold higher sensitivity than the SRY monoplex assay [25]; however, with a lower specificity. Our results with detection of fetal DNA extracted from maternal plasma emphasize the debated weaknesses of the SRY and DYS14 assays.…”

“…However, the multi-target sequence DYS14 assay was also shown to produce false-positive amplifications in some cases [12,23]. One pragmatic solution to this problem has been to set a cutoff to discriminate between true- and false-positive samples [24,25]. In contrast, detecting the SRY gene has resulted in specificities of 100%, demonstrating that false-positive results are preventable [20,21].…”