Independently of the purification method used after sequencing PCR (classical or one of the dye removal kits), we achieved the best sequencing results when the PCR products had been purified by PCR Kleen spin columns prior to sequencing PCR: 700 bases were readable, which is the maximum achievable with the ABI PRISM 310 automated sequencer.
The results of direct nucleotide sequencing depend on the quality and purity of the PCR products; there fore, the DNA purification step prior to sequencing plays the most importantrole. The purification method used for the sequencing PCR product is of secondary importance .
The use of PCR Kleen spin columns led to excellent sequencing results.
Other advantages of this method are:
A simple and rapid procedure (4 min for PCR Kleen column vs. 3 hr for gel purification)
The purified PCR product is immediately available for sequencing PCR
No exposure to ethidium bromide or UV light
It is possible to obtain sequencing results for a PCR product within 1 day
Only one specific PCR product should be present after amplification;
in cases where agarose gel electrophoresis exhibits multiple bands, purification
methods based on excisi