PCR-introduced loop structure as primer in DNA sequencing
Ronaghi, M.; Pettersson, B.; Uhlén, M.; Nyrén, P.
Biotechniques 25(5): 876
1998
ISSN/ISBN: 0736-6205 PMID: 9821590 Document Number: 487740
The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.