PUBLICATION
Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
- Authors
- Ratnayake, D., Newman, M., Lardelli, M.
- ID
- ZDB-PUB-180925-12
- Date
- 2018
- Source
- BMC research notes 11: 202 (Other)
- Registered Authors
- Lardelli, Michael, Newman, Morgan
- Keywords
- Aggregate proteins, Autophagy, Codon redundancy, Degeneracy of the genetic code, PCR based repeat region amplification, Polyglutamine repeats
- MeSH Terms
-
- Animals
- Base Sequence
- Codon/genetics*
- Embryo, Nonmammalian/metabolism
- Peptides/genetics*
- Polymerase Chain Reaction/methods*
- Repetitive Sequences, Nucleic Acid/genetics*
- Reproducibility of Results
- Trinucleotide Repeat Expansion/genetics
- Zebrafish/embryology
- Zebrafish/genetics
- PubMed
- 29587822 Full text @ BMC Res. Notes
Citation
Ratnayake, D., Newman, M., Lardelli, M. (2018) Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences. BMC research notes. 11:202.
Abstract
Objective Repeat expansion of polyglutamine tracks leads to a group of inherited human neurodegenerative disorders. Studying such repetitive sequences is required to gain insight into the pathophysiology of these diseases. PCR-based manipulation of repetitive sequences, however, is challenging due to the absence of unique primer binding sites or the generation of non-specific products.
Results We have utilised the degeneracy of the genetic code to generate a polyglutamine sequence with low repeat similarity. This strategy allowed us to use conventional PCR to generate multiple constructs with approximately defined numbers of glutamine repeats. We then used these constructs to measure the in vivo variation in autophagic degradation activity related to the different numbers of glutamine repeats, providing an example of their applicability to study repeat expansion diseases. Our simple and easily generalised method of generating low repetition DNA sequences coding for uniform stretches of amino acid residues provides a strategy for generating particular lengths of polyglutamine tracts using standard PCR and cloning protocols.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping