Figure 4

Cloning of poly(A) constructs. (A) Our basic strategy was also successfully applied to the cloning of adenine repeat sequences. Here two antiparallel oligonucleotide pairs that differ in their restriction sites and length of the poly(A) region, A₃₁ and A₃₂, were used for the initiation step. A₃₁, A₃₂, and pMK0 were digested with HindIII/BsaI, BsaI/XhoI, and HindIII/XhoI, respectively, and ligated in a single reaction to obtain pMK0-A₅₇. For elongation the BsaI/XhoI digested oligonucleotide A₃₂ was inserted into pMK0-A₅₇ cut with BsmBI and XhoI. The elongation principle is the same as described above for the Poly-Q constructs. Used restriction sites are underlined. (B) Antiparallel oligonucleotide pairs used for cloning of the poly(A) sequences. The recognition sequences and the corresponding cleavage sites of the restriction endonucleases are indicated by different colors. The inward facing BsaI sites on A₃₁ and A₃₂ were designed in a way to allow the seamless fusion of the oligonucleotides via their A-blocks. (C) Initiation and elongation step of poly(A) cloning. pMK0-A_n plasmids containing the indicated number of adenines or the empty pMK0 vector were digested with SacI (cuts 199 bp upstream of poly(A)) and XhoI. The inserts were separated on a 10% polyacrylamide (PAA) gel and visualized by ethidium bromide (EtBr) staining.