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

376 Quivy and Becker
that decreases the error rate during the amplification, and the use of an enzyme without
the 3′-5′ exonuclease activity (e.g., Vent Exo) for efficient single primer extensions.
The immobilization of the amplified fragments on paramagnetic beads exploiting the
strong streptavidin/biotin interaction is crucial for the efficiency of the sequencing
reaction because it allows the efficient removal of interfering primers from the PCR and
permits the use of a single-strand template (4,5). The attachment of the sequenced DNA
fragment to the solid support does not create a steric hindrance for the polymerase.
Frequently, the sequence of the linker oligonucleotide itself can be determined at the
very end of the genomic sequence (see Fig. 2B).
The length of sequence determined critically depends on the ability to resolve long
fragments with single nucleotide resolution, provided that the restriction site used for
linker ligation is not too close to the known sequence. Fragments of over 800 bp have
yielded reliable sequence information (Fig. 2A). Longer sequences can be obtained
in walking strategies where the newly determined sequence is in turn used to design
further reaching sets of primers. The presented strategy critically relies on the previous
identification of a suitable restriction site, ideally between 0.5 and 1 kb away from
the known sequence. Too short fragments will yield little new sequence information,
whereas large fragments (exceeding 1 kb) do not work, presumably because of
decreasing efficiencies in DNA denaturation and primer extensions reactions. Because
any kind of restriction enzyme will work, a site can be conveniently identified on a
Southern blot testing a small selection of enzymes that cut the genome at a reasonable
frequency. Alternatively, a selection of enzymes can simply be tried at random in an
LM-PCR sequencing reaction. To increase the chances that the reaction will work, the
genomic DNA may be cleaved with a whole cocktail of enzymes that collectively have
a high likelihood to produce a suitable restriction fragment.
2. Materials
2.1. Purification and Restriction of Genomic DNA
1. Suspension of nuclei from desired organism (see Note 1).
2. 0.5 M EDTA, pH 8.0.
3. RNase A, DNase free, 10 mg/mL (Boehringer, Mannheim).
4. Aqueous solution of N-lauroylsarkosine (sarkosyl), 20% (P/V) (Sigma).
5. Proteinase K, 10 mg/mL (Merck).
6. Phenol, highest quality, neutralized, and equilibrated with TE (10 mM Tris-HCl, pH 7.5;
1 mM EDTA; Aurresco).
7. Phenol/chloroform/isoamyl alcohol mixture (25241; Aurresco).
8. Chloroformisoamyl alcohol (241; Merck).
9. 0.3 M sodium acetate, pH 5.2.
10. Ethanol 100%.
11. Ethanol 80%.
12. TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.
13. Restriction enzyme with suitable 10× reaction buffer (see Note 2).
2.2. Primer Design, Primer Purification, and Annealing
of the Linker Primer (see Note 3)
The primers were synthesized on an ABI 394 DNA synthesizer and gel purified
(see Subheading 3.2.).