Document Detail

Fast, manual, nonradioactive method for DNA sequencing.
MedLine Citation:
PMID:  8353956     Owner:  NLM     Status:  MEDLINE    
We describe a protocol that allows nonradioactive detection of sequencing products after manual, direct, solid-phase sequencing of polymerase chain reaction-amplified DNA. The amplified DNA fragment to be studied is biotinylated at the 5' end of one of the two oligonucleotide primers used for amplification, allowing coupling to streptavidin-coated magnetic beads. The immobilized double-stranded DNA is then separated into single strands by alkaline treatment. A 5'-biotinylated sequencing primer is used after saturating with a biotin solution any possible remaining affinity sites on the streptavidin-coated magnetic beads. Sequencing is performed by using T7 DNA polymerase, and the sequencing products are electrophoresed in denaturing polyacrylamide sequencing gel. After transfer of the products to a nylon membrane, the sequencing pattern is revealed by chemiluminescence. Biotinylated alkaline phosphatase is bound to the 5' end of the sequencing primer via a streptavidin bridge and catalyzes the reaction by cleaving a phosphate group from a chemiluminescent substrate. The emitted photons are detected by exposing the membrane to x-ray film. This method is simple, rapid, and consistently successful and reproducible.
B Debuire; A Chabli; N Frenoy
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Clinical chemistry     Volume:  39     ISSN:  0009-9147     ISO Abbreviation:  Clin. Chem.     Publication Date:  1993 Aug 
Date Detail:
Created Date:  1993-09-23     Completed Date:  1993-09-23     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  9421549     Medline TA:  Clin Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1682-5     Citation Subset:  IM    
Service de Biochimie, Hôpital Paul Brousse, Villejuif, France.
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MeSH Terms
Base Sequence
Chemiluminescent Measurements
DNA, Neoplasm / chemistry*
DNA-Directed DNA Polymerase
Leukemia, Myelogenous, Chronic, BCR-ABL Positive / genetics
Molecular Sequence Data
Polymerase Chain Reaction
Sequence Analysis, DNA / methods*
Reg. No./Substance:
0/DNA, Neoplasm; 58-85-5/Biotin; EC 2.7.7.-/bacteriophage T7 induced DNA polymerase; EC DNA Polymerase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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