Elongase

DNA was amplified from approximately 1–20 ng of DNA as outlined in [46 (link)]. A number of PCR-based methods were used to obtain the kiwi tbx5 intron/exon boundaries required to allow primer design for moa amplification. These included: Single primer PCR - A single primer was used in a standard PCR reaction at low annealing temperatures for one cycle to allow random priming and then amplified for ~35 cycles with the annealing temperature set at the primer’s T_m. Hairpin primer ligation PCR - hairpin primers containg a PstI compatible overhang were ligated to PstI-digested DNA before standard PCRs were carried out using the hairpin primer and a tbx5-specific primer. Medium range PCR - amplification across the smaller tbx5 introns was carried out usng Elongase® (Invitrogen) or Expand Long Template PCR System (Roche) as outlined by the manufacturer. Inverse PCR - Inverse PCR was carried out to obtain intron sequences directly from moa. Biefly, moa aDNA was denatured, then dephosphorylated with shrimp alkaline phosphatase (SAP) before fresh phosphates were added with T4 Polynucleotide Kinase. The aDNA was then circularised with Circligase^TM ssDNA Ligase (Epicentre®) and subjected to rolling circle amplification using Templify^TM (Amersham). The amplified aDNA was then subjected to standard PCR using inverse primers.