Incorporation of Reporter-Labeled Nucleotides by DNA Polymerases

Abstract

The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA.

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

“…In most cases the dNTP modifications were introduced to the nucleobase. In cases where pyrimidines are employed the modifications are almost exclusively at the C5 position to avoid disturbing of Watson-Crick base pairing ability (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In addition, the C5 modification is well accommodated into the major groove of DNA without perturbing the DNA structure.…”

“…Mainly C5 alkyne modified dNTPs are used because they are accessible via metal mediated cross coupling reactions (21-23) in a straightforward manner. While many C5 modified dNTP analogs are already applied, because they are processed by DNA polymerases at least to some extent, it is still unpredictable which modification will be accepted (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) because the detailed molecular mechanism by which C5 modifications are accepted by a DNA polymerase is not at all understood. This is largely due to the absence of suitable structural data.…”

Highly Fluorescent 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐Deoxyuridine 5′‐Triphosphate as an Efficient Substrate for DNA Polymerases

“…The coupling yields are similar to those of the natural DNA synthesis. These ODNs were used for enzymatic polymerization experiments, and the synthesized sequences of ODNs are shown in Figures 1, 2 [6][7][8][9][10][11][12][13], were able to incorporate two d bt UTP into a full-length elongated DNA (22-mer); here the exception was Pfu, which has strong 3'!5' exonuclease activity. Figure S1, lanes 4 and 5, respectively), afforded better results for the elongation reactions; this might be due to stabilization of the primer/template-polymerase-triphosphate complex, but at 45 or 35 8C ( Figure S1, lanes 6 and 7, respectively) the polymerase did not work well.…”

“…Loeb et al reported that it is difficult to incorporate dNTP analogues that are modified by fluorophores such as biotin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), rhodamine (Rho), fluorescein or tetramethylrhodamine (TMR) into ODNs by using DNA polymerases that have strong 3'!5' exonuclease activity, such as Vent, Deep Vent and Pfu. [13] However, d bt UTP was found to be an excellent substrate for the enzymatic elongation reaction with KOD DNA polymerase, even though it has strong 3'!5' exonuclease activity. Since the mobility of the full-length bands (22-mer) was slightly different from that of their natural ODN counterparts in denaturing PAGE when the reactions were performed with dTTP or d bt UTP (Figure 3), the full-length band (22-mer) containing d bt U was confirmed by MALDI-TOF mass spectroscopy at m/z 7121.6 ( Figure S4).…”

“…255 8C (colored); 1 H NMR (400 MHz, CDCl 3 ): d = 9.13 (s, 1 H, H-6), 8.42 (br s, 1 H, NH), 7.39 and 7.26 (each s, each 1 H, H-4'' or H-7''), 6.43 (dd, J = 8.1, 5.9 Hz, 1 H, H-1'), 5.45 (m, 1 H, H-3'), 4.21 (m, 1 H, H-4'), 4. 8 H, O 2), 3.54 (br s, 1 H, OH-5'), 2.69-2.62 (ddd, J = 14.5, 8.1, 6.8 Hz, 1 H, H-2'a), 2.56-2.52 (dt, J = 14.5, 5.9 Hz, 1 H, H-2'b), 2.13 (s, 3 H, COCH 3 ); 13 C NMR (100 MHz, CDCl 3 ): d = 170. 6, 160.4, 157.0, 149.5, 149.1, 148.4, 145.9, 140.0, 127.8, 109.1, 103.4, 102.0, 86.6, 85.9, 77.2, 75.2, 62.3, 56.3, 56.1, 37.8, 21.0; elemental analysis calcd (%) for C 20 H 21 N 3 O 8 S: C 51.…”