The right answers to frequently asked questions
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In which applications can I use synthetic genes?
Synthetic genes might be used to adapt the codon usage for
optimising gene expression, for protein over expression and protein
engineering; as standards for Real Time PCR and PCR or to construct
hybrid genes or produce DNA vaccines. You can also create multiple
variants of a gene.
How should a gene be designed?
The desired gene coding for an artificial protein is divided into a series of 5´-end overlapping complementary oligonucleotides. To achieve maximal benefit from the gene assembly, the sequence of the oligonucleotides to be assembled has to be planned carefully with the following considerations:
- The formation of hairpins of 4 or more bases should be avoided.
- Sequences that are commonly associated with poor coupling efficiency during the chemical synthesis should be avoided.
- It is recommended to avoid sequences that introduce rare codons into a gene, if you want to achieve high levels of gene expression (protein production).
- The generation of unique restriction sites within the assembled gene will allow subsequent manipulations by recombinant DNA techniques.
Eurofins Genomics uses their proprietary software GENEius for the design of the oligonucleotides.
Are different assembly techniques possible?
Basically, there are three different approaches
for the assembly of synthetic genes. In the approach developed by
Khorana (Gupta et al., 1968) a series of sequentially overlapping
oligonucleotides are synthesised. After the annealing of the
oligos, a double stranded DNA fragment containing nicks on both
strands is formed. The nicks are sealed in a reaction with DNA
ligase, an enzyme that catalysis the formation of phosphodiester
bonds between the 5´-phosphate of one double strand oligonucleotide
fragment and the 3´-hydroxyl terminus on another adjacent double
strand oligonucleotide fragment.
An alternative strategy has been developed,
making use of the possibility of synthesising very long
oligonucleotide chains. In this approach (Rossi et al., 1982) two
oligonucleotides are constructed which upon annealing their 3´-ends
overlap. This construct is completed to a full length double strand
by a subsequent filling-in reaction using a DNA polymerase. After
polymerisation overhanging ends are generated on the double strand
fragment by digestion with an appropriate restriction enzyme.
Usually, all methods are followed by the
molecular cloning of the gene. Therefore it is necessary to
consider the cloning steps while developing the assembly strategy.
Larger genes can be divided in sub fragments that are assembled
separately. After sequence verification of the cloned sub
fragments, they are assembled to the full length construct.
Is site directed mutagenesis or de novo synthesis
If mutations are abundant and distributed across the whole gene,
we recommend de novo synthesis. De novo synthesis also gives you
the opportunity to optimise other features of the gene such as
codon usage, GC content, restriction sites etc. Site directed
mutagenesis is used if modifications are few, or are clustered in a
small part of the gene.
How can the molecular weight of a plasmid be determined?
Every bp has a specific molecular weight, in average each bp ~ 0.65 kilodaltons. Therefore, if your plasmid was 3,000 bp long, the approx. molecular weight would be 3,000 bp x 0.65 kDa per bp = 1,950 kDa.
If you have ordered a synthetic gene subcloned in our standard vector pEX-A2 (2,450 bp) and your gene is 1,000 bp long, the total length of the plasmid would be 3,450 bp and the molceular weight 2,243 kDa.
I want to use the genes or GeneStrands in qPCR. Are those products free of cross contaminations?
The synthetic genes and GeneStrands are produced on open deck robots throughout the process, therefore we cannot exclude the possibility that slight cross contamination occurs in rare cases. We have established daily cleaning and decontamination routines to make sure that we deliver the best products possible. If you want to use products from the same order in qPCR / very sensitive PCR reactions, we recommend that you contact us prior to ordering to discuss your specific needs in advance.