Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Abstract

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.

Fig. 1.

Structures of 3′-O-allyl-dCTP-allyl-bodipy-FL-510 [λ_abs(max) = 502 nm; λ_em(max) = 510 nm], 3′-O-allyl-dUTP-allyl-R6G [λ_abs(max) = 525 nm; λ_em(max) = 550 nm], 3′-O-allyl-dATP-allyl-ROX [λ_abs(max) = 585 nm; λ_em(max) = 602 nm], and 3′-O-allyl-dGTP-allyl-bodipy-650 [λ_abs(max) = 630 nm; λ_em(max) = 650 nm].

Fig. 2.

The polymerase extension scheme (Left) and MALDI-TOF MS spectra of the four consecutive extension products and their deallylated products (Right). Primer extended with 3′-O-allyl-dUTP-allyl-R6G (1), and its deallylated product 2; Product 2 extended with 3′-O-allyl-dGTP-allyl-bodipy-650 (3), and its deallylated product 4; Product 4 extended with 3′-O-allyl-dATP-allyl-ROX (5), and its deallylated product 6; Product 6 extended with 3′-O-allyl-dCTP-allyl-bodipy-FL-510 (7), and its deallylated product (8). After 30 s of incubation with the palladium/TPPTS mixture at 70°C, deallylation is complete with both the fluorophores and the 3′-O-allyl groups cleaved from the extended DNA products.

Fig. 3.

Four-color sequencing by synthesis data on a DNA chip. (A) Reaction scheme of SBS on a chip using four chemically cleavable fluorescent nucleotides. (B) The scanned four-color fluorescence images for each step of SBS on a chip: (1) incorporation of 3′-O-allyl-dGTP-allyl-Cy5; (2) cleavage of allyl-Cy5 and 3′-allyl group; (3) incorporation of 3′-O-allyl-dATP-allyl-ROX; (4) cleavage of allyl-ROX and 3′-allyl group; (5) incorporation of 3′-O-allyl-dUTP-allyl-R6G; (6) cleavage of allyl-R6G and 3′-allyl group; (7) incorporation of 3′-O-allyl-dCTP-allyl-bodipy-FL-510; (8) cleavage of allyl-bodipy-FL-510 and 3′-allyl group; images 9–25 are similarly produced. (C) A plot (four-color sequencing data) of raw fluorescence emission intensity at the four designated emission wavelength of the four chemically cleavable fluorescent nucleotides vs. the progress of sequencing extension.

Fig. 4.

Structures of 3′-O-allyl-dATP, 3′-O-allyl-dCTP, 3′-O-allyl-dGTP, and 3′-O-allyl-dTTP.

Fig. 5.

Comparison of four-color sequencing by synthesis and pyrosequencing data. (A) Four-color DNA sequencing raw data with our sequencing by synthesis chemistry using a template containing two homopolymeric regions. The individual base (A, T, C, G), the 10 repeated A's, and the five repeated A's are clearly identified. The small groups of peaks between the identified bases are fluorescent background from the DNA chip, which does not build up as the cycle continues. (B) The pyrosequencing data of the same DNA template containing the homopolymeric regions (10 T's and five T's). The first four individual bases are clearly identified. The two homopolymeric regions (10 A's) and (five A's) produce two large peaks, which are very difficult to identify the exact sequence from the data.