What is TOPO in PCR?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
What is the size of TOPO vector?
TOPO TA vector is apprx 3.9 Kb.
What is a TOPO cloning kit?
The TOPO™ TA Cloning™ Kits for Sequencing provide a highly efficient, 5-minute, one-step cloning strategy (‘TOPO™ Cloning’) for the direct insertion of Taq polymerase–amplified PCR products into a plasmid vector for sequencing.
How does the TOPO vector allow for efficient cloning?
To harness the re-ligating activity of topoisomerase I, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3′ phosphate. This enables the vectors to efficiently ligate linear DNA fragments with compatible ends. The ligation is completed in only 5 min at room temperature.
What is the purpose of TOPO cloning?
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication.
What is TA cloning used for?
Abstract. TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase.
Why is TA cloning done?
The same TA cloning vector can be used to clone any segment of PCR amplified DNA, and does not require the researcher to cut and purify the complementary vector as is done for restriction enzyme cloning. This procedure is especially useful when convenient restriction sites are not available.
Why is it called TA cloning?
The technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase, become ligated together.