'Long Distance Sequencer' Method; a Novel Strategy for Large DNA Sequencing Projects

Every DNA sequencing project involves two steps: (i) making suitable templates for all the regions to be sequenced; and (ii) running sequencing reactions and electrophoresis. The latter step can be automated by use of workstations and autosequencers. The former step requires careful experimental design and laborious DNA manipulations such as the construction of nested deletion mutants ( 1). This is often the limiting step in large sequencing projects. The ‘shot-gun’ method eliminates this complicated DNA manipulations ( 1), but many recombinant clones must be sequenced because of the random nature of this procedure. Here we describe a novel sequencing technique that utilizes recent advances in amplification of long DNA fragments by PCR ( 2). This systematic method requires minimal amount of starting DNA and eliminates complicated steps for template preparation. We have successfully used this method to determine the sequence of a cosmid insert and the genomic structure of the transforming growth factor β type II receptor gene ( 5). In our protocol, nested DNA fragments around region of interest are amplified by anchored PCR using a vectorette unit and the fragments are directly sequenced. The procedures are schematically presented in Figure 1. Amplification primers were designed in the sequenced regions of the cosmid using the MacVector program (Kodak). The following oligonucleotides, V-top 5 ′-GAAGGAGAGGACGCTGTCTGTCGAAGGTAAGGAACGGACGAGAGAAGGGAGAG-3′ and V-bottom 5′-CTCTCCCTTCTCGAATCGTAACCGTTCGTACGAGAATCGCTGTCCTCTCCTTC-3 ′ were synthesized, purified and dissolved in distilled water to a final concentration of 4 μM of each. The solution was heated at 68 C for 10 min and was slowly cooled over 30 min to room temperature to make an annealed vectorette unit (4 μM; ref. 3). V-top and V-bottom are complementary to each other except for the middle one-third, giving the vectorette unit a bubble-like structure (Fig. 1). M13 sequence-tagged 224 primer (224M13: 5 ′-TGTAAAACGACGGCCAGTCGAATCGTAACCGTTCGTACGAGAATCGCT-3 ′) (3) was phosphorylated in a 50 μl volume containing 1 × kinase buffer [70 mM Tris–HCl (pH 7.6), 10 mM MgCl 2, 5 mM dithiothreitol, 1 mM ATP], 30 μM 224M13 and 50 U T4 polynucleotide kinase. The reaction was incubated at 37 C for 30 min, heated to 68 C for 10 min then stored at –20 C. Cosmid DNA was extracted from a 1.5 ml overnight culture by an alkaline mini-prep method ( 4) into 50 μl distilled water. Two microliters of this cosmid DNA solution were enzymatically digested using AluI, BsaAI, BstUI, PalI, RsaI, AccI, AflIII, BstYI, Figure 1. Schematic representation of the method.