Mutation Detection Services
At MDS we specialize in detecting mutations such as point mutations, SNPs, insertions, deletions, and rearrangements, which may be involved in genetic disorders or disease processes. We can perform rapid mutation analysis for our clients from crude PCR reactions . Additionally, we provide PCR and sequencing services as well.
Oligonucleotide-directed mutagenesis uses a mutagenic oligonucleotide primer to alter the DNA template sequence in a defined way. Oligonucleotides can also be used to create a large number of mutations within specific regions of DNA. However, sometimes it is more efficient to do Gene Synthesis if many different mutations are desired in the same fragment of DNA.
We have the capabilities and expertise to generate many random mutations in fragments as large as 3 kb. Linker-scanning mutagenesis is used to introduce clusters of point mutations throughout a particular sequence. The polymerase chain reaction (PCR) method can be used to introduce restriction sites or point mutations into specific DNA sequences.
All samples submitted to MDS for mutagenesis will undergo an initial QC by DNA sequencing. Upon completion of the project, the introduced mutations are confirmed by sequencing, and sequences from "before and after" are compared to one another to ensure that only target mutations have been introduced. Customers are provided with plasmid DNA containing the modified gene as both purified DNA and glycerol stock, as well as all collected sequence data pertaining to their gene. At MDS, we can produce mutations at an extremely high efficiency. This service includes:
- Design of mutagenesis oligos to introduce all mutations Synthesize and purify mutagenic oligonucleotide primers
- Prepare plasmid DNA from your clones
- Perform rounds of mutagenesis via a method that will permit the use of the same vector. Each round consists of:
- Introduction and synthesis of mutagenic plasmid via high fidelity PCR.
- Enrichment for mutagenized plasmid by selective digestion of parental plasmid
- Transformation of enriched population of plasmid into E. coli.
- Miniprep DNA from 12 candidate clones containing targeted mutations (for each round)
- Sequence the plasmid DNA of the candidate clones from each round (minimum of 500 bp around the point of mutation)
- Evaluate the authenticity of clones with appropriate mutations
- The final product is provided either as a stab, purified plasmid, glycerol stock or on plates.
Random Mutagenesis: MDS can construct mutant libraries by random mutagenesis from any provided gene. Our techniques are proven for generating libraries of unexpected mutants encoding for proteins with a wide range of interesting and useful properties. Randomized DNA sequences may be cloned into any specified vector. For QC, MDS will select random clones for sequencing. Customers may choose to sequence additional clones from a given library.