DNA sequencing by synthesis using Raman and infrared spectroscopy detection

摘要

This invention provides nucleoside triphosphate analogs having the structure:;wherein B is a base and is adenine, guanine, cytosine, uracil or thymine, wherein R″ is an OH or an H, and wherein R′ is azidomethyl, a hydrocarbyl, or a substituted hydrocarbyl, and which has a Raman spectroscopy peak with wavenumber from 2000 cm−1 to 2300 cm−1 or a Fourier transform-infrared spectroscopy spectroscopy peak with wavenumber from 2000 cm−1 to 2300 cm−1, and also to methods of DNA sequencing and SNP detection.

主权项

1. A method for determining the sequence of consecutive nucleotide residues present in a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and four different nucleoside triphosphate (NTP) analogues under conditions permitting the DNA polymerase to catalyze incorporation onto the primer of a NTP analogue complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleotide residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure: wherein B is a base and is adenine, guanine, cytosine, or thymine, (ii) R′ has a predetermined Raman spectroscopy peak with wavenumber from 2000 cm−1 to 2300 cm−1 and which is different from the wavenumber of the Raman spectroscopy peak of the other three NTP analogues or R′ has a predetermined Fourier transform-infrared spectroscopy peak with wavenumber from 2000 cm−1 to 2300 cm−1 and which is different from the wavenumber of the Fourier transform-infrared spectroscopy peak of the other three NTP analogues, (iii) each of the four NTP analogues comprises a base which is different from the base of the other three NTP analogues, and (iv) R′ has the structure: wherein the wavy line indicates the point of attachment to the 3′ oxygen atom, and wherein the structure of the R′ group of each of the four NTP analogues is different from the structure of the R′ group of the remaining three NTP analogues; (b) removing NTP analogues not incorporated into the DNA extension product; (c) determining after step (b) the wavenumber of the Raman spectroscopy peak or wavenumber of the Fourier transform-infrared spectroscopy peak of the NTP analogue incorporated in step (a) so as to thereby determine the identity of the incorporated NTP analogue and thus determine the identity of the complementary nucleotide residue in the single-stranded DNA; (d) treating the incorporated nucleotide analogue under specific conditions so as to replace the R′ group thereof with an H atom thereby providing a 3′ OH group at the 3′ terminal of the DNA extension product; and (e) iteratively performing steps (a) to (d) for each nucleotide residue of the single-stranded DNA to be sequenced except that in each repeat of step (a) the NTP analogue is (i) incorporated into the DNA extension product resulting from a preceding iteration of step (a), and (ii) complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleotide residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the DNA extension product resulting from a preceding iteration of step (a), so as to form a subsequent DNA extension product, with the proviso that for the last nucleotide residue to be sequenced step (d) is optional,thereby determining the identity of each of the consecutive nucleotide residues of the single-stranded DNA so as to thereby sequence the DNA.