Automated High-Throughput Sex-Typing Assay

Abstract

BioTechniquesVol. 37, No. 4 Drug Discovery and Genomic TechnologiesOpen AccessAutomated high-throughput sex-typing assayLizhi Yu, Fernando D. Martinez & Walter T. KlimeckiLizhi YuArizona Respiratory Center, Tucson, Arizona, USA, Fernando D. MartinezArizona Respiratory Center, Tucson, Arizona, USA & Walter T. Klimecki*Address correspondence to: Walt Klimecki, Arizona Respiratory Center, P.O. Box 245030, Tucson, Arizona 85641, USA. e-mail: E-mail Address: [email protected] Respiratory Center, Tucson, Arizona, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04374DD01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Determining the sex of the donor of a human DNA sample in a rapid and accurate manner is of importance in several settings, including basic research, clinical practice, and forensics (1,2). Our group routinely performs high-throughput genotyping within large epidemiological studies. It is not uncommon to receive thousands of DNA samples, with the tube identification as the only accompanying information. These samples will then be distributed through a chain of multi-well plates, ultimately ending in 384-well PCR plates in which there is no individual identification for a given sample, only a plate identifier. In such cases, a basic test of incoming sample identification integrity and of sample processing/tracking integrity is to sex type both the original DNA sample tubes as well as the final 384-well reaction plates. These data are compared to the sex of the study participant, which is recorded in the database storing the epidemiological information, providing basic surveillance for significant sample tracking and identification problems.Recently, Gold et al. (3) published a multiplex PCR-based assay designed to produce a 475-bp fragment from the ABCD1 gene (X-chromosome) and a 231-bp fragment from the SRY gene (Y-chromosome). As the authors point out, this assay design has significant advantages, including the lack of nested primers and genomic targeting to unique, reliable sequences. In this report, we describe a significant refinement of this assay, in which these genetic regions are targeted in an end-point-based 5′-exonuclease (TaqMan®) assay. This assay takes advantage of the 5′-exonuclease activity of Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA) to cleave the reporter fluorescent molecule from the 5′ end of a probe that is hybridized 3′ to an extending PCR primer on a template strand of DNA. A key strength of this assay is that it is a homogenous reaction, requiring only one reaction setup and no subsequent reaction processing. Notably, this assay does not rely on a cumbersome electrophoretic analysis. This improvement, coupled with a 384-well reaction plate format, allows the assay to be performed in a high-throughput automated setting.Oligonucleotides were designed according to default parameters using Primer Express® software (Applied Biosystems). PCR primer and probe sequences are described in Table 1 and were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Both probes contained the Black Hole Quencher-1™ (BHQ-1) fluorescence quencher at their 3′ ends. TaqMan reactions were performed in 5 µL volumes, consisting of the following final concentrations: 900 nM of each PCR primer (of 4 primers), 200 nM of each probe (of 2 probes), 1× TaqMan Universal Master Mix (Applied Biosystems), and 5 ng/µL sample DNA. Reactions were thermal cycled in 384-well plates using a Gene-Amp® PCR System 9700 (Applied Bio-systems) at 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 58°C for 1 min. Following thermal cycling, the plates were analyzed on an ABI Prism® 7900HT Sequence Detection System (Applied Biosystems). Data were analyzed using SDS 2.0 software (Applied Biosystems). Technicians were blinded as to the actual sex of the samples prior to assigning data clusters to four categories, mal