PCR-Introduced Loop Structure as Primer in DNA Sequencing | Zendy

Mostafa Ronaghi | Zendy; Bertil Pettersson | Zendy; Mathias Uhlén | Zendy; Pål Nyrén | Zendy

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PCR-Introduced Loop Structure as Primer in DNA Sequencing

Author(s) -

Mostafa Ronaghi,

Bertil Pettersson,

Mathias Uhlén,

Pål Nyrén

Publication year - 1998

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/98255rr02

Subject(s) - primer (cosmetics) , sanger sequencing , biology , sequencing by ligation , dna sequencing , dna , primer dimer , cloning (programming) , microbiology and biotechnology , computational biology , pyrosequencing , multiple displacement amplification , library , polymerase chain reaction , genetics , genomic library , dna extraction , chemistry , multiplex polymerase chain reaction , gene , base sequence , computer science , 16s ribosomal rna , organic chemistry , programming language

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.

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