Custom Degenerated Oligo Synthesis
Degenerate oligonucleotide sequences (DOS) and PCR have been successfully used to clone cDNAs of specific proteins. Creative Biolabs relies on the reverse translation of protein subsequences to synthesize limited degenerate oligonucleotides. These products can be used as probes for Northern and Southern blot, as well as cDNA screening and genomic recombinant DNA libraries. DOS primers have also been used to amplify unknown members and homologous genes of gene families in different species.
Applications of Degenerated Oligo
Genetic analysis of a limited amount of genomic DNA can play an important role in research in the fields of justice, paleontology, genetic disease diagnosis, genetic linkage analysis, and genetic diversity. To meet our customers' scientific needs, we organically synthesize one or two purine nucleoside analogs to incorporate them into the oligonucleotide. The particular structures will be chosen to give them the character of hydrogen-bond ambiguity (or degenerate). Degenerate oligonucleotides can amplify human genomic DNA by initiating degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), increasing the number of templates for microsatellite repeat labeling genotyping. Especially for smaller genomes, DOP-PCR can amplify 200-600 times.
In summary, DOP-PCR-amplified genomic DNA is an excellent and reliable template for microsatellite genotyping and can produce noticeable bands. In all the discrete microsatellites tested, the amplification of all genomic DNA was equal, which means that the human genome was almost completely covered. Therefore, DOP-PCR seems to allow unbiased, hundreds-fold, whole-genome amplification of human genomic DNA for genotyping.
Figure 1. Amplification of cDNA using degenerate oligonucleotide sequence primers. (Cooper, 1991)
Custom Degenerated Oligo Synthesis
Creative Biolabs customizes the synthesis of degenerated oligonucleotides by using the following methods:
- The most common method for generating degenerate oligonucleotides is to use a mixed phosphoramidite at the desired position of the oligonucleotide. This procedure does not always result in expected usage of each amidite because different amidites have different coupling efficiency, and the order of addition may also bias against amidites that are added later.
- Using mixed bases. We offer oligonucleotides containing mixed or random bases that can help you create a library of binding primers that match a variable or unknown template sequence and can also be used to create diversity in cloning libraries and site-directed mutagenesis.
- Split-and-pool. Suitable for embedding diverse amino acids into other common sequences, such as CDRs in antibody variable regions.
Features
- Align multiple amino acid sequences and target a region of about 200-500 base pairs in length to achieve optimal PCR amplification.
- To increase the length of the primers (and therefore the annealing temperature) for subsequent cloning procedures, add a 5' tail (6-9 base pairs) containing restriction sites.
- The degeneracy of primers can be changed according to customer requirements to optimize the balance between primer specificity and efficiency. For example, the more degenerate the primer, the lower the specificity of annealing.
If you have any questions about our custom degenerated oligo synthesis service, you can contact us by email or send us an inquiry to find a complete solution.
Reference
- Cooper, D.L.; Baptist, E.W. (1991). Degenerate oligonucleotide sequence-directed cross-species PCR cloning of the BCP 54/ALDH 3 cDNA: priming from inverted repeats and formation of tandem primer arrays. Genome Research. 1(1): 57-62.
