Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing

Abstract

A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.

Scheme 1. Synthesis of charge-modified fluorescein-labeled ddCTP.

Scheme 1. Synthesis of charge-modified fluorescein-labeled ddCTP.

Scheme 2. Synthesis of charge-modified fluorescein-labeled ddCTP with extended linker arms.

Scheme 2. Synthesis of charge-modified fluorescein-labeled ddCTP with extended linker arms.

Scheme 3. Synthesis of charge-modified rhodamine-labeled 2′,3′-dideoxynucleotides.

Scheme 3. Synthesis of charge-modified rhodamine-labeled 2′,3′-dideoxynucleotides.

Scheme 4. Synthesis of charge-modified energy transfer (ET) terminators.

Scheme 4. Synthesis of charge-modified energy transfer (ET) terminators.

Figure 1

Electropherogram using charge-modified ddCTP (1, 4 and 5). The arrow in (A) indicates the usual starting point for the sequence data. (B) FAM–11-ddCTP (–1 charge) directly loaded on the gel after mock cycle sequencing reaction. (C) (–2 Charge) ddCTP directly loaded after mock cycle sequencing condition. (D) (–3 Charge) ddCTP directly loaded after mock cycle sequencing condition. In (D) all the dye blobs are preceding the sequencing products shown in (A). The arrows in (B–D) indicate dye blobs from unreacted dye-labeled nucleoside-5′-triphosphates and corresponding breakdown products.

Figure 2

Efficiency of incorporation of charge-modified nucleotides. TSII, Thermo Sequenase II; TSII-E681R, Thermo Sequenase II with E to R amino acid modification at position 681.

Figure 3

Efficiency of incorporation of extended linker arm, charge-modified nucleotides. L = 11, 18 and 25 indicates the number of atoms in the linker between the charged moiety and the nucleotide.

Figure 4

Electropherogram generated on an ABI Prism 377 DNA sequencer by direct loading of a sequencing reaction using two single dye terminators, FAM–25-ddCTP (7), REG–25-ddUTP (11), and two FRET terminators, FAM–TAMRA–18-ddATP (19) and FAM–ROX–18-ddCTP (20), in combination with a mutant DNA polymerase in a dGTP reaction. All the dye blobs are clearly moving ahead of the start of the sequence. The color representation is blue (C), red (T), green (A) and black (G).

Figure 5

Electropherogram showing compression relief in a dGTP dye-terminator reaction. The sequences are run in a gel containing only 4 M urea to encourage the formation of compressions.