Lecture 6 Applied Molecular Biology Molecular Evolution - high throughput · Used to create proteins with a required property that doesn't exist in nature · Or it can be used to tweak or enhance the properties of naturally existing proteins · This is just like saturation Mutagenesis o If you have structural knowledge of the protein or you know where the key residues are then use saturation mutagenesis o If there is little existing knowledge concerning the protein e.g. not possible to crystalize or too big examine by NMR spectroscopy maybe you only know the sequence use Molecular evolution o It is even possible to combine the two techniques Molecular evolutions - also known as gene shuffling, aims to mimic nature but on laboratory timescale. In essence homologous genes are 'bred' together in a PCR machine. Uses error prone PCR to deliberately introduce mistakes. There is potential to 'dope' the reaction with randomised oligonucleotides (sythentic DNAffl where some structural knowledge exists Recursive PCR - Round 1, cycle 1 Recursive PCR - Round 1, cycle 2 5 3 5 3' 5 : 3 '5' 3' 5 : 3 5 5' 3' 3 5 15' 5 3 3' 5 3 5' 5 3 5 3 5 3 5 3 3 5 5 3 :5' 3 5 3 5 D Play Recursive PCR - Round 1, cycle 3 5 3 3 D 5 5' 3 3' 5 5' 3 3 5 6 starting just 3 cycles of PCR This has created a full length gene without LIGASE But a very messy process, plenty of unproductive coupling of DNA oligos turn into a single in
For second round of PCR flanking primers used - short primers that hybridise at either end of the gene. This means that they amplify the full length gene and all products from pervious cycles are diluted Recursive PCR - Round 2 5' 3' 5 3' 3 5' 3'5' Molecular evolution · Start with a series of homologous Cut the DNA using DNAsel under strictly limiting conditions genes 3' 10000 3 . Cut the DNA using DNAse1 under strictly limiting conditions (very low conc and short time interval low tempffl 5' 5 3 3 5 5 3' 3 5' · Results in random homologous gene fragments 5 3 3 5' · Not too different from synthetic oligos from recursive PCR 5 3 3 5' · . Do recursive PCR Both rounds to get lots of recombinations of all different fragments 5' 3' 3' 5' . Remember PCR also under error prone conditions