Cutting and Splicing

Biotechnology and Recombinant DNA
Introduction	History	Cutting and Splicing	Inserting Recombinant DNA Into Cells
Selecting Transformants	Applications I Altering Phenotypes, Making Products	Applications II Study and Characterization of DNA	Ethics

The overhangs are sticky because they will hydrogen bond to another piece of DNA if it has the complementary base sequence.

If you cut two different DNA molecules with the same restriction enzyme you'll get the same (complementary) sticky ends.

The resulting restriction fragments can recombine, held together by hydrogen bonding at the complementary sticky ends.

This is how you can get DNA strands from two different molecules to recombine and produce a hybrid (or recombinant) DNA molecule.

So we can use restriction enzymes to cut DNA into manageably sized pieces, we can generate sticky ends when we do the restriction digest, and we can get DNA fragments from different sources to recombine.

Now the challenge is to insert these recombinant DNA molecules into cells. There are several techniques to insert recombinant DNA molecules into cells but the most common involve the use of a vector.

In fact, the first recombinant DNA molecule we will construct is a vector (a DNA molecule that we can insert into cells) that contains the gene or some other piece of DNA we are interested in - then we can worry about how the DNA of interest is expressed in the recipient cell.

Cutting and Splicing DNA Molecules
DNA Ligase If you want to insert a piece of new DNA (foreign DNA) into an existing strand you'd have to cut the existing strand, somehow position the foreign DNA, and then join the strands. This joining is called ligation and can be done with the enzyme DNA ligase.

Restriction Enzymes Restriction enzymes are produced by bacteria and function to protect bacteria from foreign DNA (like viruses). Restriction enzymes work by degrading foreign DNA. Restriction enzymes recognize and cut the sugar phosphate backbone of the DNA molecule at specific sites on a strand of DNA. The restriction enzyme recognition site is determined by a specific sequence of nucleotides. Thousands of restriction enzymes have been isolated, although many share the same recognition sequence. The recognition sequence for many restriction enzymes is unambiguous, that is, always the same exact bases. There are also some restriction enzymes that have ambiguous recognition sequences, for example the recognition sequence for Hinf I is a 5 base pair sequence that begins with GA and ends with TC but can have any base in the middle. We are not concerned here with the many variations and permutations, but it is helpful to know that some enzymes cut in the exact middle of their recognition site while others don't.
The restriction Sma I (isolated from Serratia marcesens) for example, recognizes the sequence CCCGGG and cuts in the middle, between the last C and the first G. This gives restriction fragments with what are called blunt ends.

If a restriction enzyme cuts somewhere other than the middle of its recognition sequence the resulting fragments will have overhangs, so called "sticky ends". In this example the restriction enzyme EcoR I (isolated from E. coli) recognizes the pattern GAATTC and cuts between the G and the A.

The overhangs are sticky because they will hydrogen bond to another piece of DNA if it has the complementary base sequence. If you cut two different DNA molecules with the same restriction enzyme you'll get the same (complementary) sticky ends. The resulting restriction fragments can recombine, held together by hydrogen bonding at the complementary sticky ends. This is how you can get DNA strands from two different molecules to recombine and produce a hybrid (or recombinant) DNA molecule.

Of course working with DNA from two different sources and looking for recombination can have its problems. There is no guarantee the restriction fragments won't recombine with themselves. But we'll get to that problem later...

So we can use restriction enzymes to cut DNA into manageably sized pieces, we can generate sticky ends when we do the restriction digest, and we can get DNA fragments from different sources to recombine. Now the challenge is to insert these recombinant DNA molecules into cells. There are several techniques to insert recombinant DNA molecules into cells but the most common involve the use of a vector. In fact, the first recombinant DNA molecule we will construct is a vector (a DNA molecule that we can insert into cells) that contains the gene or some other piece of DNA we are interested in - then we can worry about how the DNA of interest is expressed in the recipient cell.

Biotechnology and Recombinant DNA

Cutting and Splicing DNA Molecules