DNA sequence analysis

The DNA sequencing procedure is based on the dideoxynucleotide chain termination method of Sanger et al. (1977), in which nested sets (i.e., collections of single-stranded subfragments of a DNA molecule, starting at one end of the chain and extending to every position at which a particular nucleotide occurs), obtained by enzymatic synthesis, are fractionated on a high-resolution, denaturing polyacrylamide gel, from which the nucleotide sequence of the DNA molecule can be derived. The Taq Dye Deoxy Terminator Cycle Sequencing protocol (Perkin-Elmer) used in our experiments is modified compared to the original strategy at some important points. First, Taq DNA polymerase in combination with a single primer was used to prepare nested sets (Innis et al., 1988), known as cycle sequencing. Second, fluorescent tagging was used instead of radioactive labelling (Smith et al., 1985; Prober et al., 1987). Because the four dideoxynucleoside triphosphates were labelled with a differently coloured dye, the four nested sets could be generated simultaneously in a single reaction (Prober et al., 1987). In addition, a complete flexibility of choice of sequencing primer was allowed by this method.

An Applied Biosystems 373A Sequencing System (Perkin-Elmer) was used for separation and real-time detection of sequencing products, and computer interpretation of collected data.

Cfr. Protocol handbooks of ABI

Template preparation

Template DNA is prepared using the standard "Qiagen Midi (tip 100) Plasmid Purification" protocol. A 1 ml preculture at OD₆₀₀ » 0,6 is transferred to 100 ml selective LB medium. After incubation overnight, cells are collected by centrifugation and resuspended in 4 ml of buffer P1. Cells are lysed by adding 4 ml of buffer P2. 4 ml of chilled buffer P3 is added for precipitation of denatured proteins, cellular debris ...After clearing the solution by centrifugation (two times 15 min at 28.000xg), the supernatant is applied on an pre-equilibrated Qiagen-tip 100 column. After washing the column with 2 x 10 ml of buffer QC, BAC-DNA is eluted with 5 ml of buffer QF. DNA is precipitated by adding isopropanol and washed with 70% ethanol. After air-drying, DNA is redissolved in 100 µl of 10 mM Tris.HCl (pH 8,5).

A typical yield of 10 µg of BAC-DNA is obtained per Qiagen-tip 100 column. The final concentration of resuspended DNA is about 100 ng/µl, which is suitable for following sequencing reactions.

Reaction mixtures are prepared by mixing the following reagents:

Cycle sequencing is done on a TRIO-Thermoblock (Biometra) with the following program:

After cycling, sequencing products are purified using Microcon-10 spin-columns (Amicon). The reaction mixture, diluted with dH₂O up to 100 µl, is applied on a spin-column. It is concentrated by a 5 min centrifugation in a microcentrifuge at 14.000xg. After addition of another 100 µl of dH₂O, the mixture is concentrated again by centrifugation to a final volume of 20 µl. The sequencing products are recovered from the spin-column, and are precipitated with ethanol (according to the ABI recommendations). The pellet is resuspended in 2 µl loading buffer, and the complete sample is loaded onto a ABI 377 Automated DNA Sequencer using a 5 % LongRanger (FMC) gel.

Typically, one can obtain accurate gel readings of 400 bp with this protocol.

The protocol with BigDye Terminators is identical to the Dye Terminator protocol, except that the amount of template DNA can be reduced by at least a factor 2. Reaction mixtures are prepared as follows :

Thermal cycling and purification of sequencing products is identical to the protocol described above. The complete sample is loaded on the sequencing gel as described above.

In general, accurate sequences with a length of more than 600 bases can be obtained. In addition, peak height is much more uniform compared to sequences obtained with normal Dye Terminators.