Figure 1

Procedure for the sequencing of repetitive DNA.

(A) The pJAZZ linear vector was used for subcloning fragments rich in SSRs repeats. LA: left arm 10.3 kb; RA: right arm 2.2 kb. (B) Ends of fragments were sequenced using vector primers SL-1 and NZ. 3′ overhang and 5′ protruding ends were obtained by digesting the fragment with ApaI and SwaI restriction enzymes respectively. (C) Progressive unidirectional deletions produced by the digestion with exonuclease III after different times. The white box at the end of each deletion represents the portion of the fragment degraded in each consecutive interval. Note that during exonuclease treatment the vector is degraded. (D) Cloning of deletions into linear vector after purification from agarose gel and selection of a set of clones containing progressive deletions covering the size of the 7.5 kb fragment. Note that during the process fragments can be cloned in both orientations. For sequencing, SL-1 or NZ primers were used depending on the non-protected end of the deletion. Dotted lines indicate the read obtained from each clone; numbers indicate the order of assembly. (E) Obtaining the consensus sequence representing the entire 7.5 kb fragment via the assembly of ordered reads.