How does overlap extension PCR work?

How does overlap extension PCR work?

How does overlap extension PCR work?

An overlap is formed during PCR reaction. When induced polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another. This forms an overlap. When this overlap is extended by DNA polymerase yields a recombinant molecule.

How do you design primers for overlap extension PCR?


  1. Design Primers:
  2. “Extension PCR” PCR amplify the necessary fragments separately.
  3. Clean up the product using a DNA column.
  4. “Overlap PCR” Use cleaned up fragments as template in a PCR reaction:
  5. “Purification PCR” Add end primers to the Overlap PCR reaction:
  6. Gel extract the correct size fragment.

What happens during the extension stage of PCR?

Extension is achieved by using the loosened nucleotides of each base to grow the complementary DNA strand. The end result is two double-stranded products of DNA. The temperature that is used during the extension phase is dependent on the DNA polymerase that is used.

What is crossover PCR?

This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates.

How long can PCR overhangs be?

How does it work? Primers can be designed that have additional “overhang” sequence at the 3′ ends that will then be incorporated into the PCR product.

What is final extension in PCR?

Final extension evaluation The final extension step follows completion of the last PCR cycle. In this step, the PCR mixture is incubated at the extension temperature (generally 72°C) for a final 5–15 minute period.

What is the purpose of overhang in PCR?

Overhang PCR is a technique that utilizes the intrinsic fidelity of the 3′ end of primers for a specific sequence to enable you to add on more sequence to the 5′ end (see Figure 1). This allows you to use PCR to amplify a sequence whilst adding nucleotides to either the 5′ or 3′ ends of the sequence.

How do you add PCR to vector?

Experimental Procedure

  1. Run PCR and purify the PCR product: Run PCR to amplify your insert DNA.
  2. Digest your DNA:
  3. Isolate your insert and vector by gel purification:
  4. Ligate your insert into your vector:
  5. Transformation:
  6. Isolate the Finished Plasmid:
  7. Verify your Plasmid by Sequencing:

What is the purpose of overhangs in PCR?

What overhangs are in PCR?

Since two adjacent Ara-C molecules produce moderate termination, PCR products contain a mixture of 5′ overhang and blunt end DNA. Each PCR product is gel-isolated and subjected to short ligation, where cohesive end ligation is predominant.