A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis.
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A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis. / Kozina, Viviana; Capallo-Obermann, Heike; Gromoll, Jörg; Spiess, Andrej-Nikolai.
In: PLOS ONE, Vol. 6, No. 8, 8, 2011, p. 23174.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis.
AU - Kozina, Viviana
AU - Capallo-Obermann, Heike
AU - Gromoll, Jörg
AU - Spiess, Andrej-Nikolai
PY - 2011
Y1 - 2011
N2 - BACKGOUND: Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.
AB - BACKGOUND: Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.
KW - Humans
KW - Male
KW - Female
KW - Reproducibility of Results
KW - Chromosome Deletion
KW - DNA/metabolism
KW - Chromosomes, Human, Y/genetics
KW - DNA Primers/metabolism
KW - Fluorescent Dyes/metabolism
KW - Genetic Testing
KW - Multiplex Polymerase Chain Reaction/methods
KW - Nucleic Acid Denaturation/genetics
KW - Real-Time Polymerase Chain Reaction/methods
KW - Sex Chromosome Aberrations
KW - Sex Chromosome Disorders of Sex Development/genetics
KW - Humans
KW - Male
KW - Female
KW - Reproducibility of Results
KW - Chromosome Deletion
KW - DNA/metabolism
KW - Chromosomes, Human, Y/genetics
KW - DNA Primers/metabolism
KW - Fluorescent Dyes/metabolism
KW - Genetic Testing
KW - Multiplex Polymerase Chain Reaction/methods
KW - Nucleic Acid Denaturation/genetics
KW - Real-Time Polymerase Chain Reaction/methods
KW - Sex Chromosome Aberrations
KW - Sex Chromosome Disorders of Sex Development/genetics
U2 - 10.1371/journal.pone.0023174
DO - 10.1371/journal.pone.0023174
M3 - SCORING: Journal article
VL - 6
SP - 23174
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 8
M1 - 8
ER -
