DNA Assembly explained simply
DNA cloning has been around for a while, but one challenge has been the production of long stretches of nucleotide sequences. DNA assembly is a technique that allows to join multiple fragments in a single long stretch. Nowadays, multiple methods for DNA assembly exist such as Polymerase Cycling Assembly (PCA) and Gibson assembly that we will review in this article.
Polymerase Cycling Assembly
- To perform PCA it is first necessary to design the target sequence. Figure 1 shows the target sequence composed of different DNA fragment illustrated with different colors.
- The primers have to be designed with overlapping sequences according to the target design. Primers are usually 50 bp long with 20 bp overlap.
- The fragments of DNA are generated by PCR. Common size for PCR product is around 500 bp. (Figure 1. A)
- The oligo mix is melted to yield single stranded oligos. The mixture is then cooled to allow annealing of oligo dimers at their termini. (Figure 1. B)
- A polymerase with 3' exo proofreading activity uses dNTPs to fill the gaps using the complementary strand. (Figure 1. C)
- A subsequent, entirely separate polymerase step then amplifies the constructed gene by traditional Polymerase Chain Reaction (PCR).
Gibson Assembly (by Dr. Daniel Gibson in 2009) allows the production of scarless DNA constructs of multiple DNA fragments in a single, isothermic reaction. In this method, fragments and a master mix of enzymes is combined and the entire mixture is incubated at 50 °C for up to one hour.
- The first steps are the same than for PCA, the primers must be designed according to the target sequence to create overloaps.
- All fragment are digested by an exonuclease from their 5' ends resulting in single-stranded regions overlapping. (Figure 2. B)
- Fragments anneal. (Figure 2. C)
- A polymerase uses dNTPs to fill the gaps using the complementary strand. (Figure 2. D)
- A DNA ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA. (Figure 2. E)
Those methods present several advantages compared to conventional cloning:
- Faster than standard cloning schemes as it requires fewer steps
- Cheaper because it requires fewer reagents
- No scars remain between DNA fragments
- Up to 5 DNA fragments can be combined simultaneously in a single reaction (up to 15 with an optimized Gibson assembly approach)
- Can also be used for site directed mutagenesis