Ideally, the backbone will contain a variety of restriction enzyme cut sites (restriction sites) downstream of the promoter as part of a multiple cloning site (MCS). Having multiple sites allows you to easily orient your gene insert with respect to the promoter.

Restriction enzymes have two properties useful in recombinant DNA technology. First, they cut DNA into fragments of a size suitable for cloning. Second, many restriction enzymes make staggered cuts that create single-stranded sticky ends conducive to the formation of recombinant DNA.

Today restriction enzymes are an indispensable tool for biotechnology. The advantage of such enzymes is that they offer the means to very precisely cut through a double strand of DNA. Over 19,000 restrictive enzymes have been identified to-date.

Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences. In DNA cloning, restriction enzymes and DNA ligase are used to insert genes and other pieces of DNA into plasmids.

These enzymes cut both strand of the target DNA at different spots creating 3′- or 5′-overhangs of 1 to 4 nucleotides (so-called sticky ends). To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction enzymes that create compatible ends.

Opponents of cloning have concerns that technology is not yet developed enough to be safe, and that it could be prone to abuse, either in the form of clones raised as slaves, or leading to the generation of humans from whom organs and tissues would be harvested.

The cloning in human may produce certain psychological problems like psychological distress that affects the uniqueness and individuality of an organism. Moreover, it may cause certain issues in earlier or later twin’s growth.