Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences.
BMC Res Notes
; 11(1): 202, 2018 Mar 27.
Article
em En
| MEDLINE
| ID: mdl-29587822
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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Códon
/
Sequências Repetitivas de Ácido Nucleico
/
Reação em Cadeia da Polimerase
Limite:
Animals
Idioma:
En
Revista:
BMC Res Notes
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Austrália