Rapid Multiplexing and Simultaneous Detection of Human Spermatogenetic Failure with a 12 Lane Microchip Electrophoresis System

Abstract

Spermatogenic failure can result from mutations, polymorphisms, deletions, or other changes that affect one or more of the spermatogenesis-related genes, markers, or DNA sequences on the human Y chromosome, which can be investigated using a variety of different electrophoresis formats, such as heteroduplex analysis (dHPLC), single-strand conformation polymorphisms (SSCPs), denaturing gradient gel electrophoresis, or DNA sequencing.Non obstructive spermatogenic failure is most often caused by interstitial deletions on the Y chromosome, and complete deletion of the azoospermia factor (AZF) C interval of the Y chromosome is the most common known genetic cause of human male infertility. Deletions of spermatogenesis candidate genes have great utility in the diagnostic testing for male infertility. [1][2][3][4][5][6][7][8][9][10][11] Detection of microdeletions in the AZF regions (AZF A, B, C or D) is a time-consuming procedure that takes effort. Finding rapid and effective diagnostic investigation techniques is important for clinical applications and utilization in genetic detection methods in clinical settings. Multiplex PCR technology is useful in this regard. It can save time, reagents, and effort and provide information on several genes or parts of genes simultaneously in a single PCR reaction. [12][13][14] Resolving the multiplex PCR products on conventional gels or other traditional separation methods may not be helpful, especially when DNA fragment lengths are close to each other. Microchip electrophoresis technology affords the solution in such problems by offering a robust, rapid, and highly reproducible system for separation with good resolution. [15][16][17] In this report, we present an example of using combined multiplex PCR and multichannel microchip electrophoresis analysis for diagnosis of microdeletions in three Y chromosome sequence-tagged sites (STS), DYS211, DYS252 and DYS241. We illustrate how the combination of both technologies provides a rapid, effective, reproducible, and robust resolution of all fragments including those with only 11-bp differences, in the DNA range of 190-250 bp. MATERIALS AND METHODSThe Agilent 2100 Bioanalyzer microchip electrophoresis system (Agilent Technologies, Germany), which has epifluorescent detection with a semiconductor laser emitting at 630 nm was used. This Agilent chip is made from soda lime glass, has 12 sample wells with three gel-dye mix wells and one for the ladder. The depth of the channel is 10 mm, the width is 50 mm, and its effective separation length is 15 mm. Agilent microchip electrophoresis has been used for the separation of DYS252 (220 bp) and DYS241 (231 bp), (Fig. 1).The Hitachi SV 1210 Microchip electrophoresis system (Hitachi Electronics Co., Tokyo, Japan), has an LED detector (exciting wavelength at 470 nm, emission wavelength at 530 nm) and 12 channels each of which with a detecting point. Hitachi 12-lane microchips have been used for the separation of DYS211 (191 bp), DYS252 (220 bp), and DYS241 (231 bp), (Fig. 2), when the injection volt...

High resolution DNA separations using microchip electrophoresis

“…Likewise, the same instrumentation was used by Sohni et al [27] to perform routine separations for a host of PCR-amplified VNTRs markers indicative of diabetic and cardiovascular complications stemming from irregularities in several genes. A 12-lane microchip was used to detect spermatogenetic failure in a multiplexed format following an offline multiplexed PCR of three DNA sequence-tagged sites of interest on the Y chromosome [28]. With all of the required components resolved using a 1.5 cm effective separation channel, 36 samples could be processed within 180 s.…”