Human Gender Genotyping Using real-time PCR with CDY-1 Primers
Keywords:
gender genotyping, human gDNA, real-time PCRAbstract
In genotype-phenotype association study such as disease susceptibility and/or progression and drug responsibility, genomic DNA (gDNA) quantification and qualification are necessary steps prior to genotyping application. An individual DNA sample checking cannot identify miss-handling (labeling or samples mixed-up) which could especially occur from human error at any steps of gDNA preparation. Gender genotyping becomes useful for confirmation with records in participant database, leading to quality of genetic analysis results. We, therefore, developed the gender genotyping method by amplifying CDY-1 in Y-Chromosome with real-time PCR. Efficiency testing of this method with DNA samples (n = 377) showed 100% concordance with clinical data record of individual participant. This novel gender genotyping method is rapid, inexpensive, and usable to apply for genetic studies with high numbers of samples. Further development on quantitation of the amount of DNA will possibly fulfill utilization of our developed method.
References
Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001.
Glasel JA. Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratios. Biotechniques 1995; 18: 62-3.
Armbrecht M, Gloe J, Goemann W. Determination of nucleic acid concentrations using fluorescent dyes in the Eppendorf BioSpectrometer® fluorescence. In: Eppendort application note 271. Hamburg, Germany: Eppendorf AG.; 2013. p.1-10.
Vitzthum F, Geiger G, Bisswanger H, Brunner H, Bernhagen J. A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system. Anal Biochem 1999; 276: 59-64.
Piovesan A, Pelleri MC, Antonaros F, Strippoli P, Caracausi M, Vitale Lorenza, et al. On the length, weight and GC content of the human genome. BMC Res Notes 2019; 12: 106. (7 pages).
Turner S, Armstrong LL, Bradford Y, Carlson CS, Crawford DC, Crenshaw AT, et al. Quality control procedures for genome-wide association studies. Curr Protoc Hum Genet 2011; 68: 1.19.1-18.
Tischfield JA, DiCola M, Van Hummelen P, Wang Q, Brooks AI. High-throughput DNA quality control: allelic discrimination panel for determining sample contamination, gender and ethnicity in a biorepository setting. Hawaii, USA: American Society of Human Genetics; 2009.
Kayser M, de Knijff P. Improving human forensics through advances in genetics, genomics and molecular biology. Nat Rev Genet 2011; 12: 179-92.
Boonyarit H, Mahasirimongkol S, Chavalvechakul N, Aoki M, Amitani H, Hosono N, et al. Development of a SNP set for human identification: a set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population. Forensic Sci Int Genet 2014; 11: 166-73.
Simpson JL, Ljungqvist A, de la Chapelle A, Ferguson-Smith MA, Genel M, Carlson AS, et al. Gender verification in competitive sports. Sports Med 1993; 16: 305-15.
Mannucci A, Sullivan KM, Ivanov PL, Gill P. Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int J Legal Med 1994; 106: 190-3.
Aasen E, Medrano JF. Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats. Nat Biotechnol. 1990; 8: 1279-81.
Butler JM, Schoske R, Vallone PM, Kline MC, Redd AJ, Hammer MF. A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci Int 2002; 129: 10-24.
Ayub Q, Mohyuddin A, Qamar R, Mazhar K, Zerjal T, Mehdi SQ, et al. Identification and characterization of novel human Y-chromosomal microsatellites from sequence database information. Nucleic Acids Res 2000; 28: e8. (5 pages).
Gilson A, Syvanen M, Levine K, Banks J. Deer gender determination by polymerase chain reaction: validation study and application to tissues, bloodstains, and hair forensic samples from California. Calif Fish Game 1998; 84: 159-69.
Kupfer DM, Jenkins M, Burian D, Canfield DV. Use of alternative primers for gender discrimination in human forensic genotyping. [online]. 2008; [cited 2020 Jul 7]; [14 screens]. Available from: URL: https://www.faa.gov/data_research/research/med_humanfacs/oamtechreports/2000s/media/200808.pdf.
Osman J, Murad NAA, Chin SF, Jamal R. Highly sensitive and reliable human sex determination using multiplex PCR. Asia Pac J Mol Med 2014; 4: 1-4.
Kibbe WA. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 2007; 35 (Web Server issue): W43-6.
Ruamviboonsuk P, Tadarati M, Singhanetr P, Wattanapokayakit S, Kunhapan P, Wanitchanon T, et al. Genome-wide association study of neovascular age-related macular degeneration in the Thai population. J Hum Genet 2017; 62: 957-62.
Bustin S, Huggett J. qPCR primer design revisited. Biomol Detect Quantif 2017; 14: 19-28.
Choi SY, Ro H, Yi H. Designing PCR primer for cloning. In: DNA cloning: a hands-on approach. Springer Nature B.V; 2019. p. 41-43.
Mir K, Neuhaus K, Bossert M, Schober S. Short barcodes for next generation sequencing. PLoS ONE 2013; 8: e82933. (8 pages).
