Enzymatic Method for the Synthesis of Long DNA Sequences with Multiple Repeat Units

Abstract

Apolymerase chain reaction (PCR) derived method for preparing long DNA, consisting of multiple repeat units of one to ten base pairs,i sd escribed. Tw os eeding oligodeoxynucleotides,so-called oligoseeds,whichencode the repeat unit and produce ad uplex with 5'-overhangs,a re extended using athermostable archaeal DNApolymerase.Multiple rounds of heat-cool extension cycles,a kin to PCR, rapidly elongate the oligoseed. Tw enty cycles produced long DNAwith uniformly repeating sequences to over 20 kilobases (kb) in length. The polynucleotides prepared include [A] ndicating that the method is extremely flexible with regardtothe repeat length and base sequence of the initial oligoseeds.D NA of this length (20 kb % 7 mm) with strictly defined base reiterations should find use in nanomaterial applications.

Self‐Priming Enzymatic Fabrication of Multiply Modified DNA

“…The gel-electrophoresis dataf rom both extension reactions, ( Figure S2) indicated that the final DNA products consisted of ar ange of lengths, from 200 bp to 5000 bp, as expected from our previous report on this method using unmodified NTPs. [37] Further analysis of the gel band intensities, (Figure 2A-E, and Figure S7) indicated that in most cases there is minimal differ- Figure 2D. This suggests that, the size and electronegativity of bromine may have steric and/or electronic impacts on the polymerase and the higherc harged ensity and bulk of several neighbouring bromo-modifications might affect the polymerase reading and/or writing processes.O therwise we found little difference in the enzymatic processivity of different types of base modification within the same oligoseed, namely between;i )a single atom or al ong alkyl chain modification on the same base, Figure 2A,B, (cf.…”

“…The DNA products contain the modified cytidine at every fifth site on just one strand of the extended duplexa nd, in both cases the DNA produced is similar in length to the control reactionu sing the unmodified dCTP. [37] Nucleotides with more sterically demandingm odifications, 6-S-dGTP and 5acetyl-Hg-dCTP,w ere also incorporated into just one strand of the extending DNA but gave shorter products of around 500 bp which could not be further extended with additional cycling ( Figure S2 Ea nd Figure S3). Employing the same starting oligoseed but inserting either am odifiedd ATPo rd TTP results in DNA bearing 4c onsecutive modifications on the same strand.A fter 10 cycles, the methody ields DNA products bearing 7-deaza-7-I-dA (500 bp) and 5-Br-dU (5 kb) ( Figure S2 Ca nd S2 D) and furtherc ycling allows attainment of lengths of modified DNA comparable to that obtained with the native nucleotides.…”

“…Size selectionc an be simply achieved by electrophoretic separation and sample extraction using aL on-zaGel double-well system to select the desired DNA lengths. [37] For example, 250,4 50, 800 and 1200 bp samples were extracted from the dispersion of 5-I-dC bearing products,after the extension of the [A 4 G] 4 Yields of 110, 109, 206 and 199 %f or each 250, 450,8 00 and 1200 bp fraction, respectively were obtained (yields are given as ap ercentageo ft he startingo ligoseed concentration of 0.5 mm,h ence yields over 100 %a re expected). Therefore, this approachboth fabricates and amplifies modified DNA.…”

“…[17,35] Although isothermal slippage has been shown to occur for 6-10 bp repeats such as telomeric sequences, [36] we have found that intermediate sized repeat sequences (4-40 nucleotides)a re most efficiently extended by thermalc ycling methods. [37] Moreover,i ti sd ifficult to produce high copy number repeat-sequence DNA containing modified nucleotides by isothermal extension, even for short repeat sequences and low density modifications. These constraints motivated us to extend our previously reportedt hermalc ycling extension methodt of acilitate the introduction of modified bases at specificp ositions on long repeat-sequenceD NA chains, with the motivation of producing modifiedD NA for nanomaterials and diagnostic applications and potential uses in data/coding and security operations.…”

“…[38b] By using selected repeat-sequences, DNA containing single or multiple bulky modificationsa ts pecific loci on either strand can be produced. Using an adapted PCR protocol, [37] modifiedD NA with designed repeating units that include handles for furtherf unctionalization are synthesized. The heatcool cycle methode xtends DNA by relying on imperfect hybridisation within the repetitive sequence of the initial unmodified oligoseed of around 20 bases (Scheme 1A).…”