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PCR cloning method| Advantage| Disadvantages

Update time : 2020-08-04

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PCR cloning method| Advantage| Disadvantages

The difference between PCR cloningand traditional cloning is that it can amplify target DNA fragments, or even vectors, by polymerase chain reaction (PCR) and ligate them together without the use of restriction enzymes. 

PCR cloning is a fast method of cloning genes, usually used for projects that require higher throughput, and the throughput of these projects is higher than that of traditional cloning methods. It allows cloning of DNA fragments that are not available in large quantities.

Usually, a PCR reaction is performed to amplify the sequence of interest, and then it is connected to the vector by blunt-end or single-base overhang ligation before transformation. 

The early PCR cloning of Taq DNA polymerase is often used to amplify the gene. 

This results in a PCR product in which a single adenine (A) residue is added to the 3'end of the PCR product, which is independent of the template, through the normal action of the polymerase. 

These "A-tail" products are then ligated to a complementary T-tail vector using T4 DNA ligase, and then transformed.

Now, high-fidelity DNA polymerases are also commonly used to amplify sequences that do not contain 3'extension PCR products. 

The blunt-ended fragment is ligated to the plasmid vector by a typical ligation reaction or by the action of an "activated" vector containing a covalently ligated enzyme (usually topoisomerase I), which facilitates the vector:insert ligation. 

Some PCR cloning systems contain engineered "suicide" vectors that include a toxic gene to which the PCR product must be successfully ligated to allow the propagation of strains that absorb the recombinant molecule during the transformation process.

A typical shortcoming shared by many PCR cloning methods is that special vectors must be used. 

These vectors are usually sold by vendors (such as NEB) in ready-to-use linearized formats and can add significant costs to the total cost of cloning. 

Likewise, the use of specific vectors limits the researchers' choices for antibiotic resistance, promoter identity, fusion partners and other regulatory elements.

Advantage of  PCR cloning:

✧ Efficient, with dedicated carrier
✧ Suitable for high throughput

Disadvantages of  PCR cloning:

✧ Limited vector selection
✧ higher cost
✧ Lack of sequence control at the junction
Multi-fragment cloning is not easy
✧ Directional cloning is difficult