Why t4 dna ligase

Efficiently ligates DNA inserts into plasmid vectors in just 5—15 minutes at room temperature. Five products to use for insert purification, dephosphorylation, ligation and plasmid purification; includes DNA markers. A directional cloning method for protein-coding sequences that offers a rapid, efficient and high-fidelity way to transfer protein-coding regions without the need to resequence. We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems.

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Username already in use. Password Minimum of 8 characters Uppercase and lowercase letters At least one number. Confirm Password Passwords don't match. Create Account. By creating an account, you confirm that you accept the Terms and Conditions and Privacy Policy. An immobilized product can be purified through washing and released through BbsI digestion while maintaining a 4-nt overhang allowing it to be used in further ligation reactions. Each fragment is made of eight octamers and has ends complementary to the next fragment in the series.

Four groups of octamers are ligated in pooled reactions to produce bp intermediates as outlined in b. Selected intermediates are then mobilized and ligated to neighboring fragments.

A second round repeats the process to produce one fragment containing all four intermediates. In the first step, pooled ligation reactions were performed with the solid support and nine octamers. We suspected from our T4 DNA Ligase experiments that each octamer would act as a partial template for the next with the exception of the ninth octamer which would merely serve to allow the last octamer in the set of eight to ligate.

To avoid problematic regions of non-unique complementary ends found in the octamer pools, two of the pooled ligations C and D were performed in two steps, avoiding the repeated region. Each of the four products from this process, A, B, C, and D, were expected to be bp. In the second phase of construction, fragments B and D were detached from their solid support using BbsI and then ligated to the immobilized fragments, A and C, to produce fragments AB and CD.

A third digest and ligation phase, identical to the second, released the fragment CD and ligation with the immobilized AB intermediate produced the product ABCD Figure 3c. Sequencing of the bp target band verified a single product containing the bp adaptor and bp construct Figure 4b.

Sequencing of the second, smaller band revealed a product missing one of the bp intermediate fragments. The forward primer was identical to a region in the adaptor DNA attached to the first bead and the reverse was complementary to the tail end of the final intermediate to be ligated.

The first bp represent those of the adaptor while the latter bp are those of the target fragment assembled with octamers. Cost is an important consideration in gene synthesis applications. While we have not yet explored cost restriction by minimizing reaction volumes, we have shown that it is possible to undertake simultaneous oligo phosphorylation and ligation, which eliminates the need requirement of expensive pre-phosphorylated oligos. This was followed by a final BbsI digest to release the segments from their solid supports and a final ligation to join the two segments via their complementary 4-nt ends.

PCR amplication of the first segement alone, plus the first and second segments joined together, produced products of expected size Figure 5. To conclude, de novo gene synthesis offers the ability to optimize genes for unnatural hosts, alter existing or append new restriction sites, create chimeric fusion proteins, or even produce genes for completely artificial transcripts.

This approach can be more powerful and practical than conventional genetic manipulation methods [ 6 ], but its use has been limited by the high cost associated with producing error free DNA constructs from expensive and error prone oligo components [ 7 — 9 ]. In principle, a library of all possible oligos could provide a reusable stock for synthesis of many different longer DNA sequences. The present study illustrates that this approach is, in principle, possible, usingT4 DNA Ligase to combine small oligos with short overhangs.

Many DNA ligases have been isolated and characterized. While their functions are similar, their structure and cofactor dependencies vary dramatically [ 10 ] and there is dissimilar fidelity and efficiency among ligases [ 2 ]. Specifically, T4 DNA Ligase has been known for some time to be capable of joining oligos as small as pentamers and hexamers on a complete template [ 5 ], however ligases such as Tth DNA Ligase are severely inefficient at the hexamer level [ 3 ].

Our experiments suggested that T4 DNA Ligase could not efficiently catalyze the joining of an oligo hexamer to a dsDNA with a complementary overhang of three nucleotides. Binding experiments with fluorescently labeled oligos confirmed this limitation. While ligation of hexamers to 3-nt overhangs is possible with the addition of a second complementary hexamer, its low efficiency and extensive reaction time suggests DNA construction using hexamers would be extremely inefficient.

We show that octamers arranged in 4-bp overlapping frames are the next most viable alternative. Although the length of an octamer increases the size of the oligo library sixteen fold to 65, octamers provide the best potential for successive ligations while still being short enough to consider the preconstruction of an oligo library.

A reduced set of octamers that represent only selected codon combinations may yield a useful and practical library. We suspect the accuracy of the bp fragment we constructed, in the absence of any error correction, is due to the accuracy in synthesizing of short oligos and the ability of T4 DNA ligase to discriminate any base-pair mismatches that may exist. It is known that mismatches significantly reduce ligation efficiency [ 3 , 11 ]. We tested pools of up to nine unpurified octamers and showed that these could be correctly organized and combined by T4 DNA Ligase without incorporating errors into the final product.

Despite the benefits of this new approach, there are obstacles that need to be considered during selection of a set of precursor oligos.

Octamers that are complete palindromes will self-hybridize, and those with 4-nt palindromic ends will result in self-polymerization. Further, repetitive sequences longer than 8 bp cannot be synthesized from octamers, and cleavage from the solid support by BbsI digestions requires that the recognition site for this enzyme not be encoded elsewhere within the assembled sequence Thus, design is best undertaken using an algorithimic approach, to group octamers into subsets that are assembled hierarchically.

By this approach problematic sequences can often be mitigated by placing them at the junction between synthesized sub-segments. In principle, it is also possible to include in the design longer, custom oligos that span problematic sequences, although at increased cost. This present study demonstrates the feasibility of DNA synthesis by assembly of short oligo precursors. Future work will involve improving the efficiency, perhaps by adapting these methods to microfluidic devices.

Eur J Biochem. Nucleic Acids Res. Anal Biochem. The joining of short deoxyribopolynucleotides by DNA-joining enzymes. Mol Syst Biol. Biotechnol Adv. Download references. The authors thank Bonny Tam and Mauro Castellarin for assistance with sequencing, and Duane Smailus for assistance with ligation reactions.

We thank Gregg Morin for expertise, useful discussions, and advice. We thank Hamilton O. San Diego: Academic Press. Shopping Cart You have no items in your shopping cart.

Return to Previous Page. Add to Cart. Contamination with E. Fast ligation Thaw on ice and mix all reagents well. Keep all reagents and reactions on ice during setup. Single-insert ligations are optimal with an insert to vector ratio between 2 and 6. A ratio above will promote the insertion of multiple fragments, while ratios below will reduce ligation efficiency.

For difficult ligations or if the DNA concentration is unknown, it may be necessary to vary ratios and run multiple ligations. For transformation, immediately purify or dilute DNA see below.