Abstract This invention relates to the methods, apparatus, reagents and mixtures of reagents for sequencing natural or recombinant DNA and other polynucleotides. In particular, this invention relates to a method for sequencing polynucleotides based on mass spectrometry to determine which of the four bases (adenine, guanine, cytosine or thymine) is a component of the terminal nucleotide. In particular, the present invention relates to identifying the individual nucleotides by the mass of stable nuclide markers contained within either the dideoxynucleotides, the DNA primer, or the deoxynucleotide added to the primer. This invention is particularly useful in identifying specific DNA sequences in very small quantities in biological products produced by fermentation or other genetic engineering techniques. The invention is therefore useful in evaluating safety and other health concerns related to the presence of DNA in products resulting from genetic engineering techniques.
In a process for determining DNA sequence by the dideoxynucleotide chain termination method, the improvement comprising incorporating 32 S, 33 S, 34 S and 36 S in the formation of the chain terminated sequence so that a unique sulfur isotope is associated with the terminal nucleotide in the chain terminated sequence, separating the chain terminated sequences by capillary gel electrophoresis, combusting the separated chain terminated sequences to convert the incorporated sulfur to SO 2 , and determining the terminal nucleotide by measuring 64 SO 2 , 65 SO 2 , 67 SO 2 , and 68 SO 2 in a mass spectrometer thereby determining the sequence of the DNA.
Claim 2
Dependent Claim
109 words
In a process for determining DNA sequence by the dideoxynucleotide chain termination method, the improvement comprising incorporating 32 S, 33 S, 34 S and 36 S in the formation of the chain terminated sequence so that a unique sulfur isotope is associated with the terminal nucleotide in the chain terminated sequence, separating the chain terminated sequences by capillary gel electrophoresis, combusting the separated chain terminated sequences to convert the incorporated sulfur to SO 2 , and determining the terminal nucleotide by measuring 64 SO 2 , 65 SO 2 , 67 SO 2 , and 68 SO 2 in a mass spectrometer thereby determining the sequence of the DNA.
Claim 3
Dependent Claim
16 words
A process according to claim 1 wherein the isotope is incorporated only into the 2', 3'-dideoxyribonucleotides.
Claim 4
Dependent Claim
15 words
A process according to claim 1 wherein the isotope is incorporated only into the 2'-deoxyribonucleotides.
Claim 5
Dependent Claim
15 words
A process according to claim 1 wherein the isotope is incorporated into a DNA primer.