Adding Artificial Restriction Sites

Once a segment of DNA has been amplified by PCR it may be sequenced (see Ch. 24) or cloned. For cloning it is often convenient to use restriction enzymes to generate sticky ends on both insert and vector (see Ch. 22). However, it is unlikely that such sites will be located just at the ends of any particular target sequence. One way to create convenient restriction cut sites at the end of PCR fragments is to incorporate them into the primers. When designing the primers, artificial

\ Front of primer

Extra bases maCchra target form cut site

\ Front of primer

Extra bases maCchra target form cut site

FIGURE 23.10 Incorporation of Artificial Restriction Sites

Primers for PCR can be designed to have non-homologous regions at the 5' end that contain the recognition sequence for a particular restriction enzyme. After PCR, the amplified product has the restriction enzyme site at both ends. If the PCR product is digested with the restriction enzyme, this generates sticky ends that are compatible with a chosen vector.

restriction enzyme recognition sites are added at the far ends of the primers (Fig. 23.10). As long as the primer has enough bases to match its target site, adding a few extra bases at the end will not affect the PCR reaction. The bases making up the restriction site get copied and appear on the ends of all newly manufactured segments of DNA. After the PCR reaction has been run, the PCR fragment is cut with the chosen restriction enzyme to generate sticky ends. The fragment is then cloned into a convenient plasmid.

Single base overhangs may be used to clone PCR products.

Was this article helpful?

0 0

Post a comment