John M. S. Bartlett.pdf - Bio-Nica.info

Unknown Genomic Sequences 375
Fig. 2. Genomic sequences of the Drosophila hsp27 promoter region obtained following the
outlined procedure. Arrows indicate the full-length restriction fragment. (A) Long and short
gel runs of the same sequencing reaction obtained using the enzyme NruI, which cut 821 bases
upstream of the 5′-end of primer 1. (B) Upper part of a sequence obtained using the enzyme PstI
cutting 332 bases away from the 5′-end of primer 1. The sequence of the linker oligonucleotide
at the end of the genomic sequence can be unambiguously identified.
amplified by PCR using a combination of the biotinylated linker primer and primer
2 (E). The biotinylated amplification products are then immobilized on streptavidincoated
paramagnetic beads and purified from the PCR in a magnetic field (F). This
step efficiently removes unincorporated primers, which interfere with the subsequent
sequencing reaction. The immobilization also facilitates the handling of the template
fragments during the subsequent steps and specifically allows the sequencing of a singlestranded
template. The immobilized fragments are denatured, and the complementary
strand is removed by washes (G). Radioactively labeled specific primer 3, again located
3′ to primer 2 on the lower strand, now serves to prime a standard chain termination
sequencing reaction that finally generates the sequencing ladder (H). Sequences start to
be readable about 25 bases 3′ from primer 3, and may extend for up to 1 kb depending on
the location of the restriction site and the efficiency of the overall process (Fig. 2A).
Important features of the procedure are the use of a proofreading polymerase for the
PCR amplification (the Vent DNA polymerase possesses a 3′-5′ exonuclease activity)