Evagreen

Real-time duplex PCR with subsequent High Resolution Melting was performed in a total volume of 25 μL with 1 U FastStartTaq Polymerase (Roche Diagnostics GmbH), 200 μmol/L of each dNTP (Roche Diagnostics GmbH), 0.2 μmol/L of each reverse primer, 0.4 μmol/L of forward primer (S3 Table), 1 x PCR buffer (incl. 2 mmol/L MgCl₂, Roche Diagnostics GmbH), 1 x EvaGreen (Jena Bioscience) and 30–50 ng of template DNA. Amplification and analysis was done in a LightCycler 480 (Roche Diagnostics GmbH) using filter set 465–510 with an initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s with a single measurement at the end of each cycle and a final elongation at 72°C for 7 min. Subsequently, High Resolution Melting was done at 95°C for 1 min (4.4°C/s), 40°C for 1 min (2.2°C/s), 70°C for 1 s (1°C/s), an increase to 90°C with the continuous acquisition mode (0.02°C/s), and 40°C for 1 s (2.2°C/s). The samples were genotyped by using the Melt Curve Genotyping Analysis Module of the LightCycler 480 Software (release 1.5.0 SP3, Roche Diagnostics GmbH). When calculating the first negative derivative, the wild-type amplicon and the deletion specific amplicon appeared as clearly distinguishable melting peaks with a T_m at 80.5°C and 79.0°C, respectively (S1 Fig).

Deceased donor DNA was extracted from lymphocytes used for the pre-transplantation cross match test as part of routine practice.
Each amplification reaction was carried out in a total volume of 25 μL 10 mMTris-HCl buffer pH 8.4 containing 50 mM KCl, 0.2 mM of each dNTP, 2 μM MgCl₂, 0.4 μM of each primer (CAV1F: TGGTATCTAACATACAGCC and CAV1R: GGAGGTATGGCATGTGGA), 200 ng DNA, and 0.6 U Taq DNA polymerase (Life Technologies, Carlsbad, CA, USA). After an initial denaturation step at 94°C for 3 min, 35 cycles of 1 min at 94°C, 1 min of hybridization at 60°C, and 1 min of extension at 72°C were carried out. A final extension period of 7 min was performed at 72°C. Size and specificity of PCR fragments were controlled on 1% agarose gels after incorporation of an intercalator (EvaGreen, Jena Bioscience, Jena, Germany).
After purification with the ExoSap-IT enzyme (USB) (Affymetrix, Santa Clara, CA, USA), amplicon nucleotide sequences were determined using an automated DNA sequencer (ABI Prism® 3130 Genetic Analyser, Life Technologies, Carlsbad, CA, USA). Fragments were amplified with the CAV1F and CAV1R primers, labeled with the BigDye® Terminator v3.1 kit (Life Technologies, Carlsbad, CA, USA) and analyzed with SeqScape v2.5.6 software (Life Technologies, Carlsbad, CA, USA).

Real-time duplex PCR with subsequent melting curve analysis was performed as shown for HH5 with 1 μmol/L of insertion specific primers, 0.5 μmol/L of wild-type specific primers (S3 Table), 1 x PCR buffer (incl. MgCl₂, Roche Diagnostics GmbH), 1 x EvaGreen (Jena Bioscience) and 30–50 ng of template DNA. Amplification and analysis was done in a LightCycler 480 (Roche Diagnostics GmbH) using filter set 465–510 with an initial denaturation at 95°C for 10 min, followed by 33 cycles at 95°C for 15 s, 57°C for 20 s, and 72°C for 30 s with a single measurement at the end of each cycle at 80°C. Subsequently a melting curve was recorded from 80°C to 95°C (5 acquisitions/C°). Melting curves were then evaluated as described for HH5. Each of the two amplicons showed a clear distinct melting peak with a T_m at 86.6°C and 88.7°C for the wild-type and insertion specific amplicon, respectively (S2 Fig).
In addition to verify the insertion size, long range PCRs were performed with DNA from affected and unaffected cattle using primers targeting the up- and downstream insertion sides and spanning the insertion sides with ~ 100bp distance each, using the Expand Long Range PCR plus kit (Roche Diagnostics). Agarose gel electrophoresis was performed on samples from affected and unaffected cattle to further characterize the insertion.