Ultrafast Diagnosis of the Genetic-related Disorders Using the Combined Technologies of Multiplex PCR and Multichannel Microchip Electrophoresis

Jabasini, Mohammad; Ewis, Ashraf A.; Xu, Feng; Ping, Guichen; Fouad, Maged; Shinka, Toshikatsu; Nakahori, Yutaka; Ishikawa, Mitsuru; Baba, Yoshinobu

doi:10.2116/analsci.21.1537

Cited by 4 publications

(2 citation statements)

References 13 publications

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“…The positive results obtained from these studies paved the way for a wide use of microchip electrophoresis in several DNA research studies. Moreover, the CE technology found several applications in DNA analysis by either using the chip technology alone or in combination with other technologies such as polymerase chain reaction (PCR), where the PCR is performed for the DNA markers then followed by microchip separation [ 23 ].…”

Section: Resultsmentioning

confidence: 99%

Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

et al. 2016

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We investigated an anomalous DNA separation where two DNA fragments from the human Y-chromosome sY638 (64 bp) and sY592 (65 bp), with only one base pair difference, were separated. This result is abnormal since in a previous study, we found that 5 bp was the minimum difference between two DNA fragments that the microchip electrophoresis system can separate. The formation of a mini-loop in the structure of the DNA fragment of sY638 (64 bp) was strongly expected to be the reason. To investigate this, we synthesized three modified DNA fragments for sY638 (64 bp), and the modifications were in two expected locations for possible mini-loop formation. Later, the separation between sY592 (65 bp) and the three modified fragments of sY638 (64 bp) was not possible. Thus, we conclude that the formation of a mini-loop in the structure of the DNA is the reason behind this anomalous separation.

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Section: Resultsmentioning

confidence: 99%

Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

et al. 2016

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“…Microchip electrophoresis as an ultrafast and reliable tool that can be utilized in screening assays for the diagnosis of presymptomatic disease states and detection of the underlying causes of non obstructive azoospermia and hypo spermatogenesis, i.e., spermatogenetic failure. [18][19][20] Approximately 20% of men who seek help at infertility clinics present with non obstructive oligo or azoospermia. Most of these men are chromosomally normal but may have microdeletions in the AZF regions of their Y chromosomes, which cause them to have low sperm counts or severe forms of spermatogenic failure.…”

Section: Resultsmentioning

confidence: 99%

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

Jabasini

Ewis

Fouad

et al. 2006

Biological & Pharmaceutical Bulletin

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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...

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Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection

Lin¹,

Chang²,

Wu³

et al. 2010

Biosensors and Bioelectronics

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Ultrafast Diagnosis of the Genetic-related Disorders Using the Combined Technologies of Multiplex PCR and Multichannel Microchip Electrophoresis

Cited by 4 publications

References 13 publications

Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

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

Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection

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