4-3.2.3. Incorporation of cDNA into a vector

The blunt-ended cDNA termini are modified in order to ligate into a vector to prepare ds-cDNA for cloning. Since blunt-end ligation is inefficient, short restriction-site linkers are first ligated to both ends.

Linker

It is a double-stranded DNA segment with a recognition site for a particular restriction enzyme. It is 10-12 base pairs long prepared by hybridizing chemically synthesized complementary oligonucleotides. The blunt ended ds-DNAs are ligated with the linkers by the DNA ligase from T4 Bacteriophage.

Figure 4-3.2.3.1: Modification of cDNA termini using linkers.