An approach to the use of stable isotopes for DNA sequencing.
The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5' end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.
Jacobson
K B
KB
Biology Research Division, Oak Ridge National Laboratory, Tennessee 37831.
Arlinghaus
H F
HF
Schmitt
H W
HW
Sachleben
R A
RA
Brown
G M
GM
Thonnard
N
N
Sloop
F V
FV
Foote
R S
RS
Larimer
F W
FW
Woychik
R P
RP
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds; D005821:Genetic Techniques; D007503:Iron Isotopes; D013058:Mass Spectrometry; D000075163:Metallocenes; D009942:Organometallic Compounds; D016133:Polymerase Chain Reaction; D011108:Polymers
D005296:Ferrous Compounds; D007503:Iron Isotopes; D000075163:Metallocenes; D009942:Organometallic Compounds; D011108:Polymers; C032949:ferrocenecarboxylic acid; D004247:DNA; C004998:ferrocene