In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR
Figure 1
The DNA construct of the Fab fragments for mRNA display. (a) From the 5′ end it consists of a T7 promoter (T7), ribosomal-binding site (RBS), variable region and constant region of the H chain or L chain, epitope tag, and a poly A tail. The H chain epitope tag is a Myc tag and the L chain is a FLAG tag. The poly A tail is for purification of mRNA-displayed molecules by biotin-oligo dT in combination with streptavidin beads. (Middle) Details of the linkable 5′ UTR. Between the T7 promoter (boxed) and RBS (green), 21 bases from the beginning of T7 promoter transcription start point (arrow), in other words +1 to +21 of the H chain DNA (red) and L chain DNA (orange) are designed so that they overlap with each other during overlap-extension PCR. (b) The reverse-transcribed DNA strands overlap at the overlapping region (red) to form an H chain gene and L-chain-gene-linked DNA.
