Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency
Genet. mol. res. (Online)
; 5(2): 323-332, 2006. ilus, graf, tab
Article
en En
| LILACS
| ID: lil-442566
Biblioteca responsable:
BR1.1
ABSTRACT
An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
LILACS
Asunto principal:
Enfermedades de los Bovinos
/
Análisis de Secuencia de ADN
/
Deficiencia del Factor XI
/
Tamización de Portadores Genéticos
Tipo de estudio:
Guideline
/
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Genet. mol. res. (Online)
Asunto de la revista:
BIOLOGIA MOLECULAR
/
GENETICA
Año:
2006
Tipo del documento:
Article
País de afiliación:
India