Direct DNA Sequencing of Complementary DNA Amplified by the Polymerase Chain Reaction

Protocols for the sequence analysis of conventional single-stranded or double-stranded DNA templates are often unsuitable for the direct sequencing of DNA fragments generated by the polymerase chain reaction (PCR) (1 ,2 ). The features that can distinguish PCR products as templates for sequencing include (a) contamination of the reactions by nonspecific PCR amplification products that are complementary to the sequencing primer, (b) the persistence of “leftover” PCR primers from the amplification reactions, and (c) the potential for competition between one strand of the amplified fragment and the oligonucleotide used for the sequencing. The various approaches that have been used to overcome these problems include

1.	The use of 5′-end-labeled DNA-sequencing primers that are complementary to regions between the PCR primers (3 );
2.	Gel purification of amplified DNA to remove unwanted fragments and primer (4 );
3.	Spin columns for the separation of leftover primers from high mol wt material (5 , 6 );
4.	“Asymmetric” or “unbalanced” PCR priming to generate an excess of single strands during the initial amplification (7 );
5.	Addition of dimethylsulfoxide (DMSO) to sequencing reactions with short annealing times (8 ); and
6.	The use of several short, high-temperature, sequencing cycles (9 ).