US 5,002,868 A · DNA sequencing process using stable isotopes

Abstract

A DNA sequencing process using specific stable isotopes associated with specific terminators for labels. The process includes a step of incorporating a stable isotope in at least one of the deoxynucleoside triphosphates and/or the dideoxynucleoside triphosphates such that a terminated strand has included within it or at the end a stable isotope such as an isotope of sulphur. Replicated strands are then separated by performing gel electrophoresis thereon. The location of the DNA strand with the stable isotope assigned to a terminator is analyzed preferably by resonance ionization spectroscopy. The stable isotopes can be chosen such that specific labels are assigned to at least one, and preferably to each base, in the dideoxynucleoside triphosphates. In the preferred embodiment, each of the bases (A, T, G and C) are associated with a specific stable isotopic label, and can be analyzed in a single track which enhances the accuracy of the sequencing process.

Claims9

Dependent claims

A DNA sequencing process using individual stable isotopes that are associated with individual deoxynucleotides and/or terminators comprising the steps of:

introducing template DNA strands with primer hybridization into a solution containing deoxynucleoside triphosphates (dNTP) having thymine, adenine, cytosine, and guanine bases;
DNA polymerase;
buffer;
magnesium ion;
and dideoxynucleoside triphosphate (ddNTP) wherein the ratio of dNTP to ddNTP is selected, said ddNTPs causing replication termination when incorporated into the replicated strand as dideoxynucleotide at a frequency dependent upon the ratio of dNTP to ddNTP;
incorporating a specific stable isotope into at least one of the dNTPs and or ddNTPs such that a terminated strand has a specific stable isotope associated with each terminator at its end;
separating the terminated strands by performing gel electrophoresis thereof;
and
determining where each said specific stable isotope is located on the gel.

6 more elements

Dependencies are inferred from the claim text.

DNA sequencing process using stable isotopes

Life of this patent

DNA sequencing process using stable isotopes

Life of this patent

Abstract

Claims9