Figure 1

Design for the isolation of probe from the tiGHR I cDNA and construction of competitor fragment to use in quantitative PCR. a Diagram of probe amplification from the tiGHR I cDNA using degenerate oligonucleotide B and E. ECD, extracellular domain; TMD, transmembrane domain; ICD, intracellular domain; black box 1, conserved N glycosilation site of the GHR extracellular domain (LNWTLLNI); black box 2, coding sequences corresponding to the proline-rich site in the intracellular domain box I (PKIKGIDP); Probe, 458-bp DNA fragment of tiGHR I obtained with the B–E oligonucleotide mixes and cloned in Easy T-vector (shadow boxes); Competitor, 473 DNA fragment obtained from two subcloning steps to produce a tandem of two copies of the Pst I–Acc I fragment of the Probe; I and J, internal oligonucleotide from the probe fragment designed for the amplification of target and competitor; E, EcoR I; P, Pst I; A, Acc I; S, Sac I. b 2% agarose gel image with the PCR amplification products using oligonucleotides I and J: 1, PCR negative control (without template); 2, PCR using a RT reaction from tilapia liver mRNA as template; 3, PCR using competitor sequence as template, 4, PCR using probe as template.