Well, #GoldenGateCloning surely has opened a few gateways to reliable molecular cloning in #molecularbiology. It is an innovative modification of the classical dig-lig cloning method with a smart use of #TypeIIS restriction enzymes and #T4DNALigase. Here's a crisp undernote!
Principle
The principle of #GoldenGateCloning lies in the difference of #TypeIIS restriction enzymes like #BsaI, #BpiI, #BsmBI etc. from typical restriction enzymes, which is that they cleave outside of their recognition sequence, creating four base flanking non-palindromic #overhangs. With about 256 possible overhang sequences, multiple fragments of DNA (upto 5 with <15% yield) can be assembled by using combinations of overhang sequences.
Protocol
Fig.: A simplified scheme of #GoldenGateAssembly.
The reaction setup is simple as any digestion or ligation reaction with occasional use of additives like #BSA and extra #ATP.
Thermal cycling depends on the number of fragments, but generally looks as below:
Merits
The prime merit of the protocol is the absence of scars (#scarless) between assembly fragments.
Although the original destination vector and digested fragment may spontaneously religate in a one pot reaction, this transient construct retains functional Type IIS sites and will be re-digested. In contrast, formation of the desired ligation product is #irreversible because this construct does not retain the enzyme recognition sites. As a result, the ligation process is close to #100percentEfficient.
The fragment-specific overhang sequences allow specific and respective #assembly of multiple fragments at one go.
Time #efficient and less costly as #DigestionLigation is coupled in a #SingleTube, withdrawing the hassles of gel purification and quantification.
There is no #BufferIncompatibility issues, as single restriction enzyme is used.
Demerits
As the process is #irreversible, subcloning to a different vector is not possible.
There may be one or several BsaI sites in the insert fragment.
Applications
Cloning of multiple fragments.
Site directed mutagenesis.
More clues here:
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