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T/A CLONING

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T/A CLONING

  1. 1. T/A CLONING
  2. 2.  TA cloning is a subcloning technique that doesnt use restriction enzymes and is easier and quicker than traditional subcloning. The technique relies on the ability of adenine (A) and thymine (T) (complementary base pairs) on different DNA fragments to hybridize and, in the presence of ligase become ligated together. PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3 end of the product
  3. 3.  Such PCR amplified inserts are cloned into linearized vectors that have complementary 3 thymine overhangs. Commercialized kits with pre-prepared vectors and PCR reagents are currently sold, greatly speeding up the process.
  4. 4. PROCEDURE Creating the insertInsert is created by PCR using Taq DNAPolymerase.This polymerase lacks 3 to 5 proofreadingactivity and, with a high probability, adds a single, 3-adenine overhang to each end of the PCR product. It is best if the PCR primers have guanine at the 5 end as this maximizes probability of Taq DNA polymerase adding the terminal adenosine overhang.
  5. 5.  Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity should not be used as they do not leave the 3´ adenine-overhangs
  6. 6. CREATING THE VECTOR The target vector is linearized and cut with a blunt-end restriction enzyme This vector is then tailed with ddTTP using terminal transferase. (ddTTP to ensure the addition of only one T residue) This tailing leaves the vector with a single 3- overhanging thymine residue on each blunt end.
  7. 7. DIAGRAM OF TA CLONING
  8. 8. ADVANTAGES OF TA CLONING No need of restriction enzymes other than generating the linearized vector. Procedure is simple and fast as compared to traditional way of cloning. TA cloning can be used where no restriction sites can be used in traditional cloning.
  9. 9. DRAWBACK The major problem is that the gene has a 50% chance of getting cloned in the reverse direction. The method also needs DNA ligase.

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