Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers

 

Sequencing double stranded DNA templates has become a common and efficient procedure (10) for rapidly obtaining sequence data while avoiding preparation of single stranded DNA. Double stranded templates of cDNAs containing long poly(A) tracts are difficult to sequence with vector primers which anneal downstream of the poly(A) tail. Sequencing with these primers results in a long poly(T) ladder followed by a sequence which is difficult to read. In an attempt to solve this problem we synthesized three primers which contain (dT)17 and either (dA) or (dC) or (dG) at the 3' end. We reasoned that the presence of these three
bases at the 3' end would 'anchor' the primers at the upstream end of the poly(A) tail and allow sequencing of the region immediately upstream of the poly(A) region.

Using this protocol, over 300 bp of readable sequence could be obtained. We have applied this approach to several other poly(A)-containing cDNA clones with similar results. Sequencing of the opposite strand of these cDNAs using insert-specific primers occurred directly upstream of the poly(A) region. The ability to directly obtain sequence immediately upstream from the poly(A) tail of cDNAs should be of particular importance to large scale efforts to generate sequence-tagged sites (STSs) (11) from cDNAs (12,13).

Protocol

1. Synthesize anchored poly (dT)17 with anchors of (dA) or (dC) or (dG) at the 3' end on a DNA synthesizer and use after purification on Oligonucleotide Purification Cartridges.

2. For sequencing with anchored primers, denature 5-10 mg of plasmid DNA in a total volume of 50 ml containing 0.2 M sodium hydroxide and 0.16 mM EDTA by incubation at 65oC for 10 minutes.

3. Add the three poly(dT) anchored primers (2 pmol of each) and immediately place the mixture on ice. Neutralize the solution by adding 5 ml of 5 M ammonium acetate pH 7.0.

4. Precipitate the DNA by adding 150 ml of cold 95% ethanol and wash the pellet twice with cold 70% ethanol.

5. Dry the pellet for 5 minutes and then resuspend in MOPS-Acid buffer.

6. Anneal the primers by heating the solution for 2 minutes at 65oC followed by slow cooling to room temperature for 15-30 minutes.

7. Perform sequencing reactions, using modified T7 DNA polymerase and a-[32P]dATP (> 1000 Ci/mmol) using the protocol described earlier.