PCR Cloning | PCR In Vitro Cloning | PCR Based Cloning | Gene Cloning Using PCR |
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PCR cloning is a rapid
PCR cloning is a rapid method for cloning genes, and is often used for projects that require higher throughput than traditional cloning methods can accommodate. It allows for the cloning of DNA fragments that are not available in large amounts.
In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest.
Typically, a PCR reaction is performed to amplify the sequence of interest, ....
PCR cloning often usesTaq DNA Polymerase to amplify the gene. This results in a PCR product with a single template-independent base addition of an adenine (A) residue to the 3' end of the PCR product, through the normal action of the polymerase.
These "A-tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase, followed by transformation.
High-fidelity DNA polymerases are also now routinely used to amplify sequences with the PCR product containing no 3' extensions. The blunt-end fragments are joined to a plasmid vector through a typical ligation reaction or by the action of an "activated" vector that contains a covalently attached enzyme, typically Topoisomerse I, which facilitates the vector:insert joining.
In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest.
Typically, a PCR reaction is performed to amplify the sequence of interest, ....
PCR cloning often usesTaq DNA Polymerase to amplify the gene. This results in a PCR product with a single template-independent base addition of an adenine (A) residue to the 3' end of the PCR product, through the normal action of the polymerase.
These "A-tailed" products are then ligated to a complementary T-tailed vector using T4 DNA ligase, followed by transformation.
High-fidelity DNA polymerases are also now routinely used to amplify sequences with the PCR product containing no 3' extensions. The blunt-end fragments are joined to a plasmid vector through a typical ligation reaction or by the action of an "activated" vector that contains a covalently attached enzyme, typically Topoisomerse I, which facilitates the vector:insert joining.
3 سال پیش
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