Structural Biochemistry/Directed Changes in DNA

Proteins with new functions can be created through directed changes in DNA. In the classic genetic approach, mutations are generated randomly throughout the genome. Analysis of mutatants reveal which genes are altered and DNA sequencing identifies the exact changes. Now, utilizing recombinant DNA technology, specific mutations can be made in vitro. New genes can be constructed by three changes to the sequence: deletions, insertions, and substitutions.

Deletions
Deletions occur when one or multiple base pairs are cleaved from a DNA sequence. A specific deletion can be produced by cleaving a plasmid at two sites with a restriction enzyme which removes a large segment of DNA. A smaller deletion can be made by cutting a plasmid at a one site and the linear DNA can be digested by an exonuclease that removes nucleotides from both strands. T

Deletion is when a base pair is removed from the DNA sequence. This causes the DNA strand to be shorter.

Substitutions
Single amino acid substitutions can be produced by oligonuleotide-directed mutagenesis. This mutation can be made if (1) a plasmid containing the gene or cDNA for the protein is obtained and (2) the base sequence around to site to be altered is known. If serine is to be changed to cystine, the code TCT needs to be changed to TGT, a point mutation. The key to this mutation is to prepare an oligonucleotide primer that is complementary to this region except that it contains TGT instead of TCT. The mismatch of one base pair out of 15 does not make a large difference. Replication of these DNA strands with the mutated primers leads to two kinds of plasmids, one with the original sequence and one with the mutated sequence.

A substitution occurs when one base pair is replaced with another. There are three main effects of substitutions in DNA:

1. The substituted base could encode a stop codon. This will stop the DNA from producing the rest of the amino acids and could lead to a nonfunctioning protein that might be harmful to the biological system or damaging to the DNA.

2. The new substituted base could encode a different amino-acid, which will make the DNA produce a different amino-acid than it was supposed to.

3. Silent Mutation- This is when the changed base still codes for the same amino acid. These mutations are rarely harmful to the DNA or biological system.

Insertions
Insertions of a DNA sequence can be made using cassette mutagenesis. In this technique, plasmid DNA is cut with a pair of restriction enzymes to remove a short segment which can be replaced by a synthetic double stranded oligonucleotide.

Insertions are the result of extra bases added into the DNA sequence.

Frame Shifts
This is caused by insertions and deletions. Deletions will make the DNA sequence shorter, while insertions will extend the DNA sequence’s length. Codons are encoded in groups of 3 bases. A deletion or insertion of bases will shift the whole sequence, and the DNA could potentially encode a whole new set of amino-acids.