Enzymatic assembly of overlapping DNA fragments

Abstract

Three methods for assembling multiple, overlapping DNA molecules are described. Each method shares the same basic approach: (i) an exonuclease removes nucleotides from the ends of double-stranded (ds) DNA molecules, exposing complementary single-stranded (ss) DNA overhangs that are specifically annealed; (ii) the ssDNA gaps of the joined molecules are filled in by DNA polymerase, and the nicks are covalently sealed by DNA ligase. The first method employs the 3'-exonuclease activity of T4 DNA polymerase (T4 pol), Taq DNA polymerase (Taq pol), and Taq DNA ligase (Taq lig) in a two-step thermocycled reaction. The second method uses 3'-exonuclease III (ExoIII), antibody-bound Taq pol, and Taq lig in a one-step thermocycled reaction. The third method employs 5'-T5 exonuclease, Phusion® DNA polymerase, and Taq lig in a one-step isothermal reaction and can be used to assemble both ssDNA and dsDNA. These assembly methods can be used to seamlessly construct synthetic and natural genes, genetic pathways, and entire genomes and could be very useful for molecular engineering tools.

Figure 15.1

Assembly vector primer design. (A) A linear DNA sequence that is to be assembled into a vector. The first and last 40 bp of DNA sequence is underlined. (B) Two primers that could be used to PCR-amplify pUC19 to produce a vector containing overlaps to the sequence shown in (A), thus producing a circle. The primer sequences include regions that can anneal to pUC19 (nonbolded, lowercase), NotI restriction sites (bolded and italicized, uppercase) to release the insert from the vector, and 40-bp overlaps (underlined, uppercase) to the ends of the DNA sequence shown in (A).

Figure 15.2

Two-step thermocycled in vitro recombination. Two adjacent DNA fragments (magenta and green) sharing terminal sequence overlaps (thickened black line) are joined into one covalently sealed molecule by a two-step thermocycled reaction.

Figure 15.3

One-step thermocycled assembly of overlapping DNA segments. Two adjacent DNA fragments sharing terminal sequence overlaps are joined into one covalently sealed molecule by a one-step thermocycled reaction.

Figure 15.4

One-step isothermal assembly of overlapping DNA fragments. Two adjacent DNA fragments sharing terminal sequence overlaps are joined into one covalently sealed molecule by a one-step isothermal reaction.

Figure 15.6

Overlapping oligonucleotide design for assembly into pUC19. (A) A 340-bp sequence, which includes 20 bp overlapping sequence to PCR-amplified pUC19 (nonbolded lowercase) and NotI restriction sites (bolded and underlined). Because 56 bp is used for assembly into and release from pUC19, only 284 bp of unique sequence (uppercase) is synthesized. (B) The sequence in (A) can be synthesized from the eight 60-mer oligos shown, which contain 20 bp overlaps.

Figure 15.5

Isothermal assembly of overlapping oligonucleotides into pUC19. Eight 60-base oligos (red lines) are directly assembled into pUC19 (gray lines), in vitro, to produce a dsDNA fragment. N indicates the NotI restriction site (black line), which is added to release the fragment from the pUC19 vector.