Solid phase amplification

Solid phase amplification (SPA) was performed as a special form of arrayed on-chip amplification using directly surface-bound primers forming a very dense carpet like probes in microarray applications (Bing DH et al., 1996; Adessi C et al., 2000; Nickisch-Rosenegk M et al., 2005; Fedurco M et al., 2006). Amplification can occur via two processes. First “interfacial amplification”, where freely diffusing DNA target molecules attach to surface- bound primers, primers are elongated to complementary DNA copies and these ssDNA molecules stay attached to the surface, while the initial DNA molecule returns to the solution after the annealing step. Second “surface amplification”, where the free end of the attached ssDNA copy hybridizes to a sequence-complementary surface-attached primer in immediate proximity. This time primer elongation leads to building bridges between primers, as both elongated DNA molecules stay attached to the surface. SPA thus leads to the generation of a colony of molecules attached to the surface and located in the same region. Synthesized PCR

products can be visualized either by confocal microscopy or fluorescence microscopy using Cy5-dye fluorescence of modified primers, or the fluorescence of intercalating dyes.

Spot array design. For solid phase amplification applications, 3 different surface-bound primer-pairs were spotted on reaction center B (∅ 500 µm) of LOC chips (chapter 2.3.1). Primer-pairs were spotted in a 2x2 spot array structure using a Nadelspotter (spotting operations were performed by Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany). The following array design was spotted: the array consisted of 4 spots, while each spot comprised a different primer-pair content. On one spot primer-pairs DY-fw and DY-rv for amplifying male locus DYS392 were spotted, on a second spot primer-pairs DX-fw and DX-rv for amplifying female locus DXS10134, on a third spot primer-pairs AM-fw and AM- rv for amplifying human amelogenin as a positive control and on the forth spot primers DY- fw and DX-fw as negative control, providing no complementary sequences after amplification. Primer sequences were listed in the appendix, chapter 9.2. 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. Solid phase amplification PCR reactions. For performing solid phase amplification on LOC chips the QuantiFast SYBR Green I PCR kit was used for 2-step PCR (QIAGEN GmbH, Hilden, Germany). Several PCR protocols were tested as described in the following, first only “SPA PCR” using genomic DNA and second a “combined PCR setup” using preamplified PCR products. Before PCR, array-spotted LOC chips were preheated for 15 min at 95°C for adapting the material to hot temperatures (chapter 2.3.1).

Using “SPA PCR”, 1 µl of total reaction mix contained 0.5 µl of 2x QuantiFast SYBR Green I PCR Master Mix (final 1x), 0.3 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany) and 0.2 µl of 10x concentrated input DNA. For input DNA 5 ng/µl of male or female reference DNA was used, resulting in a final concentration of 1 ng reference DNA present in 1 µl total reaction volume. 1 µl of prepared master mix was placed on primer- array spotted reaction center B of LOC chips and was immediately covered with 5 µl of Sealing Solution to prevent evaporation and external cross-contamination. 2-step SPA PCR cycling conditions were 5 min initial denaturation at 95°C, followed by 35 cycles of 95°C for 30 sec and 60°C for 60 sec.

Using the “combined PCR setup”, preamplified PCR products were used as input DNA. For preamplification the QuantiTect SYBR Green I PCR kit was used for 3-step PCR (QIAGEN GmbH, Hilden, Germany), while 10 µl of total PCR reaction mix contained 5 µl of 2x QuantiTect SYBR Green I PCR Master Mix (final 1x), 1 µl of 10 µM primer solutions (DYS392-fw and DYS392-rv or DXS10134-fw and DXS10134-rv or Amel1 and Amel2; final 1 µM per primer), 2 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany) and 1 µl of 10x concentrated input DNA. For input DNA 5 ng/µl of male and female reference DNA was used, resulting in a final concentration of 500 pg reference DNA present in 10 µl total reaction volume. Primer sequences were listed in the appendix, chapter 9.2. Reactions were performed in sterile 0.2 ml PCR-tubes (Eppendorf AG, Hamburg, Germany) using a conventional in-tube PCR thermocycler (advanced Primus 96, PeqLab Biotechnologie GmbH, Erlangen, Germany). 3-step PCR cycling conditions were 15 min initial denaturation at 95°C, followed by 35 cycles of 94°C for 30 sec, 55°C for 60 sec, 72°C for 30 sec, and final product extension at 72°C for 7 min. PCR products were analyzed on polyacrylamide gels (CleanGel 10%, ETC GmbH, Kirchentellinsfurt, Germany) and subsequent DNA silver staining (DNA silver staining kit, GE Healthcare, Uppsala, Sweden) (chapter 2.2.3). Blank and negative controls were included in every reaction batch. For 0.1 µl input DNA in the following “combined PCR setup”, 0.033 µl of each preamplified PCR product tube DYS392, DXS10134, Amel for male or female DNA was utilized. The “combined PCR setup” comprised three reaction steps performed consecutively on LOC chips, which were summarized in table 2, while the QuantiFast SYBR Green I PCR kit was used for 2-step PCR (QIAGEN GmbH, Hilden, Germany).

Table 2. Combined PCR setup. Interfacial amplification, surface amplification and a hybridization step were

performed consecutively on the LOC chip’s surface for optimizing solid phase amplification output.

Order of performance Composition of reaction mix Thermal cycling conditions

1. Interfacial amplification

1 µl total reaction mix contained 0.5 µl of 2x QuantiFast SYBR Green I PCR Master Mix (final 1x), 0.4 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany) and 0.1 µl of preamplified PCR products.

2-step PCR protocol: 5 min initial denaturation at 95°C, followed by 30 cycles of 95°C for 30 sec and 55°C for 60 sec.

2. Surface amplification

1 µl total reaction mix contained 0.5 µl of 2x QuantiFast SYBR Green I PCR Master Mix (final 1x) and 0.5 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany).

2-step PCR protocol: 5 min initial denaturation at 95°C, followed by 30 cycles of 95°C for 30 sec and 55°C for 60 sec.

3. Hybridization

1 µl total reaction mix contained 0.5 µl of 2x QuantiFast SYBR Green I PCR Master Mix (final 1x) and 0.5 µl of preamplified PCR products.

Hybridization protocol: 3 min denaturation at 95°C, followed by 40°C

hybridization temperature for 30-60 min.

Washing protocol and detection. After finishing “SPA PCR” or “combined PCR setup”, Sealing Solution was washed away from reaction center B using sterile water and array chips were subjected to the washing protocol described in table 1. Washing procedures removed the Sealing Solution efficiently as well as unbound PCR remainings. LOC chips were washed manually by applying a 10 µl overlay of buffers onto the SPA 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). 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 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). Additionally, fluorescence signal were detected using the lab-on-a-chip integrated Fluorescence Reader and appropriate software “Norbert.VI” for manual picture taking (LabVIEW 8.6, National Instruments Germany GmbH, Munich, Germany). Pictures were taken with exposure times of 1000 ms, 2000 ms and 4000 ms at room temperature as well as at 40°C and 60°C. Furthermore, to check for detached primers and PCR products in the SPA reaction solution, reaction mixes were analyzed on polyacrylamide gels (CleanGel 10%, ETC GmbH, Kirchentellinsfurt, Germany) and subsequent DNA silver staining (DNA silver staining kit, GE Healthcare, Uppsala, Sweden) (chapter 2.2.3).