Pyrosequencing protocol requiring a unique biotinylated primer.
BACKGROUND
DNA sequencing has markedly changed the nature of biomedical research. Large-scale sequencing projects have generated several millions of potential polymorphisms widespread in the human genome requiring validation and incorporation into screening panels. As a consequence, high-throughput analysis of these variants in different populations of interest is now the cornerstone of structural genomics. Pyrosequencing is a versatile technique allowing an easy 96-well typing format. However, every polymorphism requires a specific labeled primer to generate a single-stranded DNA fragment containing the region of interest.


METHODS
We describe how with an adjusted primer stoichiometry we can standardize the labeling of every amplicon with a single biotinylated universal primer (BM13S).


RESULTS
We circumvent the need for specific biotinylated primers for each single-nucleotide polymorphism (SNP) under study. As an example, we assessed this novel protocol by genotyping three SNPs mapping calpain-10, caveolin-1 and CYP19A1.


CONCLUSIONS
The present approach represents an alternative to standard pyrosequencing protocols, since it requires a single biotinylated primer that is suitable for each SNP under study.
Royo
Jose Luis
JL
Departamento de Genomica Estructural, Neocodex SL, Sevilla, Spain. jlroyo@neocodex.es
Pascual
Manuel Hidalgo
MH
Salinas
Ana
A
Tello
Francisco Jose
FJ
Rivero
Maria Del Carmen
Mdel C
Herrero
Eduardo Ferrero
EF
Real
Luis Miguel
LM
Ruiz
Agustín
A
D019921:Biotinylation; D017931:DNA Primers / Q000737:chemistry*; D023281:Genomics / Q000379:methods; D006801:Humans; D008722:Methods; D009154:Mutation; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers