Microarray hybridization

In microarray applications biological probes are arrayed onto planar surfaces and slides through the use of a robotic array spotter, while biological probes comprise short single stranded DNA oligonucleotides. PCR-amplified DNA fragments in solution are subjected to hybridization with these surface coupled complementary strands, providing information on the corresponding nucleotide sequence (Schena M, 1999). Hybridization happens specifically

via the building of hydrogen bonds between complementary sequences of probes and amplified DNA fragments. Due to the small size, a huge amount of probes detecting for thousands of gene fragments can be arrayed, just dependent on the size of the supporting surface. As fluorescently tagged primers were utilized, e.g. tagged with chromogenic dyes “Cy3” or “Cy5”, PCR products are fluorescently labeled as well. Detection of hybridization events between surface-bound probes and primer-labeled fluorescent PCR products is performed via on-line detection methods.

Spot array design. A 2x2 spot array was designed for the determination of PCR-amplified male and female sample material, according to gender determining approaches used in forensic research. Probes of the microarray were designed in a way, to detect a 6 bp insertion sequence AAAGTG between male and female PCR-amplified amelogenin fragments. So either probes matched the 106 bp X-chromosomal sequence or were complementary to the 112 bp Y-chromosomal sequence. Three different probes were designed for hybridization providing distinct sites for specific PCR product hybridization. Probes Amelo1(Y) and Amelo3(Y) were designed for binding male amelogenin PCR products having the 6 bp insert, while probe Amelo2(X) was destined for hybridizing to female PCR products lacking the 6 bp insert (probe sequences were listed in the appendix, chapter 9.2). Successful hybridization events of fluorescently labeled PCR products were detected via the intensity of fluorescence signals.

For microarray applications, probes were spotted on reaction sites (∅ 1.6 mm) of a multi LV- PCR microdevice (AmpliGrid AG480F, Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany; chapter 2.3.1) and on reaction center B (∅ 500 µm) of LOC chips (chapter 2.3.1). Probes were spotted in a 2x2 array structure using a Nadelspotter (spotting operations were performed by Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany). There were 6 different array designs spotted. Each array consisted of 4 spots, while full arrays were spotted with 4 spots having the same probe content (array designs 1-3), and split arrays where only 2 spots had the same probe content (array designs 4-6). Array designs were as follows: (1) 4 spots of Amelo1(Y), (2) 4 spots of Amelo2(X), (3) 4 spots of Amelo3(Y), (4) 2 spots of Amelo1(Y) + 2 spots of Amelo2(X), (5) 2 spots of Amelo2(X) + 2 spots of Amelo3(Y), (6) 2 spots of Amelo1(Y) + 2 spots of Amelo3(Y). Spot size was about 120 µm (100-160 µm) with a spot distance of about 160 µm, comprising an array diameter of about 400 µm. Spotting solution contained 50 µM oligonucleotide probe solution dissolved in 1x Advalytix spotting buffer AT100. Spotting, washing and passivation steps after spotting were done by Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany as well.

PCR and hybridization of male and female human genomic reference DNA. For PCR amplification prior to hybridization and for detection of hybridized PCR products, a Cy3 fluorescently labeled primer Amel1-f-Cy3 was used in combination with an unlabeled primer Amel2. Primer sequences were listed in the appendix, chapter 9.2. For amplification and hybridization, the QIAGEN Fast Cycling PCR kit (QIAGEN GmbH, Hilden, Germany) was used for 3-step PCR. 1 µl total reaction volume contained 0.5 µl of 2x QIAGEN Fast Cycling PCR Master Mix (final 1x), 0.2 µl of 5x QIAGENQ-Solution (final 1x), 0.1 µl of 10 µM primer solutions Amel1-f-Cy3 and Amel2 (final 1 µM per primer) and 0.1 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany) for negative controls or 0.1 µl of 10x concentrated input DNA. For input DNA concentrations of 1 ng male or female reference DNA was used, resulting in a final concentration of 100 pg present in 1 µl total reaction mix. 1 µl of prepared master mix was placed on spotted reaction center B of LOC chips or on spotted reaction sites of a multi LV-PCR microdevice and was immediately covered with 5 µl of Sealing Solution to prevent evaporation and external cross-contamination. 3-step PCR cycling was performed according to the manufacturer’s protocol: 5 min initial denaturation at 95°C, 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, and final product extension at 72°C for 1 min. PCR performance was subsequently followed by a hybridization protocol comprising 3 min denaturation at 95°C and 40°C hybridization for 30-60 min.

Washing protocol. After hybridization, Sealing Solution was washed away from reaction sites using sterile water and array chips were subjected to the following washing protocol (originally provided by Alopex (Kulmbach, Germany) for using the “Chromo Chip System”) comprising washing solutions Wash 1 and Wash 2 (table 1) (washing solutions were taken from the Medical Genetic Center, Munich). Washing procedures removed the Sealing Solution efficiently as well as unbound PCR products.

Table 1. Washing protocol and compositions of washing buffers Wash 1 and Wash 2 after PCR and array

hybridization. Buffers Wash 1A and Wash 2 needed to be autoclaved at 120 °C for 20 min prior to use. Washing procedures were performed at room temperature.

Washing solutions Composition Concentrated Buffer Solutions

1.0 l Wash 1 (working solution) 100 ml Wash 1A (10x) 20 ml Wash 1B (5x) 880 ml H2Odest. Wash 1A (10x) = 3 M NaCl 0.3 M Na3 Citrate 2H2O pH 7.0 Wash 1B (5x) = 10% (w/v) Natriumdodecylsulfate (SDS) 1.0 l Wash 2 (working solution) 100 ml Wash 2 (10x) 900 ml H2odest. Wash 2 (10x) = 0.3 M NaCl 30 mM Na3 Citrate 2H2O pH 7.0 Washing protocol 5x 1 min Wash 1 2x 1 min Wash 2 1x 3 min Wash 2

For washing the multi LV-PCR microdevice an automated washing station appropriate for these slides was utilized (AdvaWash, Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany). LOC chips were washed manually by applying a 10 µl overlay of buffers onto the hybridization reaction center B of the chips. After washing, array chips were dried for 5 min at 37°C until slide surfaces were totally dry and stored in darkness for the scanning process (max. 3 h).

Detection. The multi LV-PCR microdevice was scanned for hybridized PCR products using an automated PMT laser-based microarray scanner system (ProScanArray Microarray Analysis System, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) and appropriate software (ProScanArray Scanner Software, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA). LOC chips were scanned using an inverted optical microscope (Axio Obsever.Z1, Carl Zeiss GmbH, Jena, Germany) with integrated fluorescence unit for excitation and appropriate filters for excitation, emission and detection. Excitation was done using a HBO 100 high-pressure mercury lamp (HBO 100, Leistungselektronik JENA GmbH, Jena, Germany). Due to the Stokes-transition between the absorption and emission spectrum, it is possible to separate the bright excitation light from the weak fluorescence light in the light path of the microscope via using appropriate filter sets. Pictures of fluorescence intensities were taken using a CCD camera (Rolera-XR, QImaging, Surrey BC, Canada) and QCapture Pro 6.0 imaging software (QImaging, Surrey BC, Canada).