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

374 Quivy and Becker
Fig. 1. The use of Linker Tag Selection LM-PCR for genomic dideoxy sequencing. Bold
lines denote known sequences, and thin lines the unknown sequences to be determined. The
arrows and dotted lines indicate primer extension reactions. R: Strategic restriction site. P1, P2,
P3, and LP stand for the primers 1, 2, 3, and the linker primer, respectively. The biotin moiety on
the linker is represented by a filled circle, and the streptavidin-coated paramagnetic beads by the
shaded boxes. (H): Radiolabeled P3 is used to prime dideoxy sequencing reactions.
The steps of the reaction are outlined in Fig. 1. A restriction enzyme is selected
that cleaves the genomic DNA somewhere within the unknown sequence but within
1 kb from the known sequence for which the specific primers have been designed.
Cleavage creates a defined end (A). The cleaved genomic DNA is denatured, and
the gene-specific primer 1 is annealed to the known sequence and extended by a
polymerase until the end of the restriction fragment (B). This creates a blunt end to
which a short double-stranded linker is ligated (C). The linker DNA consists of two
complementary oligonucleotides, the longer one being biotinylated at its 5′-end. After
denaturation, the specific primer 2, representing sequences more 3′ from primer 1 on
the lower strand of the known sequence, is then annealed to the upper strand, which
now carries the biotinylated linker oligonucleotide at its 5′-end. The primer is again
extended to the end (D) creating a double-stranded fragment that contains a region
of unknown DNA flanked by known sequences. The genomic sequences can now be