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. 2003 Aug 15;31(16):4769-78.
doi: 10.1093/nar/gkg669.

Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing

Patrick J Finn  1 , Matthew G Bull, Haiguang Xiao, Paula D Phillips, John R Nelson, Greg Grossmann, Satyam Nampalli, Bernard F McArdle, J Anthony Mamone, Parke K Flick, Carl W Fuller, Shiv Kumar
Affiliations

Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing

Patrick J Finn et al. Nucleic Acids Res. .

Abstract

A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

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Figures

Figure 1
Figure 1
Incorporation efficiency of dye-labeled oligo-lysine and oligo- trimethyllysine modified dideoxynucleoside-5′-triphosphates by Thermo Sequenase II.
Figure 2
Figure 2
Direct load DNA sequence using oligo-lysine modified dideoxynucleoside-5′-triphosphate terminators 6, 7, 14 and 15. The color representation is blue (C), red (T), green (A) and black (G).
Figure 3
Figure 3
Effect on resolution versus read length using charge-modified terminators 7 and 21 in a single color sequencing reaction.
Figure 4
Figure 4
Effect of different charge carriers on resolution using charge modified terminators 7 and 21 in a single color sequencing reaction.
Figure 5
Figure 5
Direct load DNA sequence using oligo-trimethyllysine-modified dideoxynucleoside-5′-triphosphate terminators 20, 21, 28 and 29. The color representation is blue (C), red (T), green (A) and black (G).
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Scheme 1. Synthesis of single dye-labeled oligo-lysine modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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Scheme 1. Synthesis of single dye-labeled oligo-lysine modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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Scheme 2. Synthesis of FRET labeled oligo-lysine modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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Scheme 2. Synthesis of FRET labeled oligo-lysine modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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Scheme 3. Synthesis of single dye-labeled oligo-trimethyllysine-modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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Scheme 3. Synthesis of single dye-labeled oligo-trimethyllysine-modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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Scheme 4. Synthesis of FRET labeled oligo-trimethyllysine modified 2′, 3′-dideoxynucleoside-5′-triphosphates.
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