Difference between seamless cloning and homologous recombination

There is no difference. According to the information about seamless cloning and homologous recombination, there is no difference between seamless cloning and homologous recombination.

1. Seamless cloning, also known as homologous recombination, allows single to multiple fragments to be inserted into a vector at the same time, without the need for multiple digestion and purification. Usually, seamless cloning can complete the ligation of multiple fragments within 5-15 min, and its cloning positive rate is up to 98% or more.

2. When we perform PCR amplification before seamless cloning, there should be a homologous sequence of 15-20 bases between vector and fragment, and between fragment and fragment, why? This is because there are three enzymes in our seamless cloning kit, namely T5 exonuclease, DNA polymerase and DNA ligase. T5 exonuclease has the activity of a 5'-3' exonuclease, which can degrade the sequence from 5'-3', exposing the vector and the 3' homologous sequence of the fragment, and after base complementary pairing, the DNA polymerase adds the missing base to the DNA polymerase. After base complementary pairing, DNA polymerase adds the missing base and then DNA ligase connects the fragment to the vector, thus achieving the function of vector recombination. Therefore, without the addition of the homologous sequence, there is no way to connect the vector to the fragment or the fragment to the fragment.