Rapid sequencing of DNA based on single molecule detection

Conference · Tue Jan 01 00:00:00 EST 1991

OSTI ID:6250905

Soper, S A; Davis, L M; Fairfield, F R; Hammond, M L; Harger, C A; Jett, J H; Keller, R A; Marrone, B L; Martin, J C; Nutter, H L; Shera, E B; Simpson, D J

Sequencing the human genome is a major undertaking considering the large number of nucleotides present in the genome and the slow methods currently available to perform the task. We have recently reported on a scheme to sequence DNA rapidly using a non-gel based technique. The concept is based upon the incorporation of fluorescently labeled nucleotides into a strand of DNA, isolation and manipulation of a labeled DNA fragment and the detection of single nucleotides using ultra-sensitive laser-induced fluorescence detection following their cleavage from the fragment. Detection of individual fluorophores in the liquid phase was accomplished with time-gated detection following pulsed-laser excitation. The photon bursts from individual rhodamine 6G (R6G) molecules travelling through a laser beam have been observed as have bursts from single fluorescently modified nucleotides. Using two different biotinylated nucleotides as a model system for fluorescently labeled nucleotides, we have observed synthesis of the complementary copy of M13 bacteriophage. Work with fluorescently labeled nucleotides is underway. We have observed and manipulated individual molecules of DNA attached to a microbead with an epifluorescence microscope. 16 refs., 3 figs., 1 tab.

• Research Organization: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
• Sponsoring Organization: DOE/ER
• DOE Contract Number: W-7405-ENG-36
• OSTI ID: 6250905
• Report Number(s): LA-UR-91-562; CONF-910123-16; ON: DE91008591
• Resource Relation: Conference: OE/LASE '91: 4th Society of Photo-Optical Instrumentation Engineers (SPIE) international symposium, Los Angeles, CA (USA), 20-25 Jan 1991
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
47 OTHER INSTRUMENTATION
MAN
DNA SEQUENCING
NUCLEOTIDES
LABELLING
BACTERIOPHAGES
DNA-ASE
FLUORESCEIN
FLUORESCENCE
LABELLED COMPOUNDS
LASERS
MICROSCOPY
MOLECULAR BIOLOGY
PHYCOBILINS
RHODAMINES
AMINES
ANIMALS
AROMATICS
CARBOXYLIC ACIDS
DYES
ENZYMES
ESTERASES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROXY ACIDS
HYDROXY COMPOUNDS
LUMINESCENCE
MAMMALS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
PARASITES
PHENOLS
PHOSPHODIESTERASES
PIGMENTS
POLYPHENOLS
PRIMATES
REAGENTS
STRUCTURAL CHEMICAL ANALYSIS
VERTEBRATES
VIRUSES
550200* - Biochemistry
550400 - Genetics
440600 - Optical Instrumentation- (1990-)