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Using software and robotics equipment to reduce manual workload when creating multi-mutation protein variants by SOE PCR

IP.com Disclosure Number: IPCOM000243442D
Publication Date: 2015-Sep-22

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Using software and robotics equipment to reduce manual workload when creating multi-mutation protein variants by SOE PCR.

Splicing with overlap extension PCR (SOE-PCR) can be used to introduce several mutations into a gene of interest [1,2]. We have developed a method to do this in a semi-automated way, using a computer program that creates the primers and pipetting lists, which can then be used by a liquid handler robot or for hand pipetting.

5x fragment PCR

Assembly

Step 1 - The backbone DNA sequences (templates)

For this example, two random DNA sequences have been generated. They are slightly different, which is a common situation when one has two or more similar backbones, in which one wants to introduce mutations. Metadata about CDS and mature regions has been added as Feature descriptions. This file is saved as "DNA.embl"

ID EMBOSS_001; SV 1; linear; unassigned DNA; STD; UNC; 300 BP.
AC Backbone1
XX
FT CDS 59..235
FT mat_peptide 107..235
XX
SQ Sequence 300 BP; 83 A; 67 C; 75 G; 75 T; 0 other

caacccgaca aggcgccgcc actcctagga ggtatatggg tcctgtgtcc gcttaaaaat 60

gcagcggata gtcgtcctcg gtatatttta tgggaacaag aaccttagcg ttgcggtaat 120

agctagagcg gtcatggatg ttggagaggg agaggcttac ccatttcaag tcgccaacat 180

ttattcagat gctgccgtat acagggaact gagttaccat tttaataggt acaattgatc 240

cccaattagc agcagattag gtccacaata cttcatggtc tagacggaaa tgccaactcg 300
//

ID EMBOSS_002; SV 1; linear; unassigned DNA; STD; UNC; 300 BP.

AC Backbone2
XX
FT CDS 59..235
FT mat_peptide 107..235
XX
SQ Sequence 300 BP; 83 A; 67 C; 75 G; 75 T; 0 other

caacccgaca aggcgccgcc actcctagga ggtatatggg tcctgtgtcc gcttaaaaat 60

gcagcggata gtcgtcctcg gtatatttta tgggaacaag aaccttagcg ttgcggtaat 120

agctagtcat gagcgggatg ttggagaggg agaggcttac ccatttcaag tcgccaacat 180

ttattcagat gctgccgtat acagggaact gagttaccat tttaataggt acaattgatc 240

cccaattagc agcagattag gtccacaata cttcatggtc tagacggaaa tgccaactcg 300
//

The corresponding protein sequences (CDS and mature) would be:

>CDS1 [59 - 235]
MQRIVVLGIFYGNKNLSVAVIARAVMDVGEGEAYPFQVANIYSDAAVYRELSYHFNRYN
>Backbone1 [107 - 235]
SVAVIARAVMDVGEGEAYPFQVANIYSDAAVYRELSYHFNRYN



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>CDS2 [59 - 235]
MQRIVVLGIFYGNKNLSVAVIASHERDVGEGEAYPFQVANIYSDAAVYRELSYHFNRYN
>Backbone2 [107 - 235]
SVAVIASHERDVGEGEAYPFQVANIYSDAAVYRELSYHFNRYN

Step 2 - Design variants

A text file with the desired variants is created, one variant per line is created. The format is

mutation 1,mutation 2, mutation 3; backbone

In this example 13 variants are designed, with up to 3 mutations, and the same mutations are generated in both the backbones, ie. a total of 26 variants. However, the mutations do not necessarily need to be the same in both backbones. The variant list is saved to "variants.txt"

A3K;Backbone1
I5V;Backbone1
A6D;Backbone1
*6aA;Backbone1
A6*;Backbone1
A3K,I5V;Backbone1
A3K,I5V,*6aA;Backbone1
I5V,A6*;Backbone1
L35N;Backbone1
Y37H;Backbone1
H38D;Backbone1
A3K,Y37H;Backbone1
I5V,Y37H;Backbone1
A3K;Backbone2
I5V;Backbone2
A6D;Backbone2
*6aA;Backbone2
A6*;Backbone2
A3K,I5V;Backbone2
A3K,I5V,*6aA;Backb...