engleski

A one-step multiplex PCR without downstream amplicon analysis for screening Y-chromosomal microdeletions

INTRODUCTION AND AIM Worldwide, 10-15% of couples suffer from infertility and in half of these cases defective spermatogenesis is the reason. Y-chromosomal microdeletions (YCMD) as a genetic cause are found in 7.3% of the infertile men. The long arm of the Y-chromosome contains three different regions (AZFa, AZFb, AZFc) [1] with different prevalences within the clinical manifestation of YCMD: the AZFc region has the highest with 79%, in contrast to AZFb (9%) and AZFa (3%). Deletions can be attributed to single regions but may also be encountered in two or three regions simultaneously (AZFb+c: 6%, AZFa+b+c: 3% ) [2]. The testicular phenotype in patients with YCMD can differ substantially: men with deleted AZFa or AZFb+c regions exhibit Sertoli cell-only syndrom (SCO) and thus azoospermia, while AZFc deletions result in a more heterogeneous clinical and histological phenotype from oligozoospermia to azoospermia A plethora of methods exist that investigate YCMD based on conventional or real-time multiplex PCR (polymerase chain reaction). All of these methods have the drawback that the resulting amplicons must be checked for validity (correct size) by using agarose or capillary electrophoresis. As this is a rather time- and money-consuming step, we aimed to develop a multiplex PCR that can identify correct amplicons by using the melting peaks from a melting curve program that is conducted directly after amplification. MATERIAL AND METHODS 31 different genomic DNA's (isolated from testicular biopsies) containg different AZF deletions were used to develop the real-time multiplex PCR. Concentrations of DNA's were checked on a Nanodrop spectrophotometer and amplifications performed on a Roche LightCycler using EvaGreen chemistry and different combinations of classical and newly designed primers. The validity of amplicons was checked on a 2100 Bioanalyzer (Agilent). General appliccability of the new method was additionally tested on a plate-based Stratagene MX-3000 platform. RESULTS We have developed two sets of 4-plex primers that are able to distinguish the amplification of established regions within the AZF locus and that are designed on maximizing the peak resolution of the resulting amplicons. Each set contains one amplicon for the AZFa, AZFb and AZFc region and a control gene (sry or amelogenin). The melting peaks of these amplicons differ at least 2°C so that single amplicons can be identified within the melting curve containing four peaks, thereby avoiding ‘peak-fusion’. Using the novel DNA-binding dye EvaGreen instead of classical SybrGreen improves the peak resolution substantially. Great effort was made in titrating the primers in the multiplex setup in a way that established equal peak sizes for all amplicons, eliminating the effect of different PCR efficiencies. CONCLUSION Our newly developed method of combined real-time amplification and subsequent melting curve analysis significantly shortens and simplifies the process of testing patients for YCMDs without losing the accuracy or precision of established methods. By using an improved DNA-binding dye (EvaGreen) and specific primer combinations that maximize the peak resolution, we developed a real-time PCR based method for detecting AZFa, AZFb and AZFc deletions that eliminated further validation of the amplicons. A more detailed analysis is needed to correlate morphology with deletions (especially thickness of lamina propria).

Y-chromosomal microdeletions; real-time PCR; multiplex PCR; EvaGreen; melting curve analysis

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