Document Detail


Fast, manual, nonradioactive method for DNA sequencing.
MedLine Citation:
PMID:  8353956     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
B Debuire; A Chabli; N Frenoy
Related Documents :
8038696 - The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of s...
22387026 - Histone h3 lysine 56 methylation regulates dna replication through its interaction with...
11679316 - Identification by subtractive hybridization of sequences specific for salmonella enteri...
7859166 - Rapd reactions from crude plant dna. adding rnase a as a "helper enzyme".
8190636 - A simple in vivo footprinting method to examine dna-protein interactions over the yeast...
10730196 - Cisplatin.
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    
Affiliation:
Service de Biochimie, Hôpital Paul Brousse, Villejuif, France.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Base Sequence
Biotin
Chemiluminescent Measurements
DNA, Neoplasm / chemistry*
DNA-Directed DNA Polymerase
Humans
Leukemia, Myelogenous, Chronic, BCR-ABL Positive / genetics
Magnetics
Microspheres
Molecular Sequence Data
Polymerase Chain Reaction
Sequence Analysis, DNA / methods*
Chemical
Reg. No./Substance:
0/DNA, Neoplasm; 58-85-5/Biotin; EC 2.7.7.-/bacteriophage T7 induced DNA polymerase; EC 2.7.7.7/DNA-Directed DNA Polymerase

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


Previous Document:  Estimation of plasma and urinary hemoglobin by a rate spectrophotometric method.
Next Document:  Rapid multicomponent analysis of hemoglobin derivatives for controlled antidotal use of methemoglobi...