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Hexaprimer amplification refractory mutation system PCR for simultaneous single-tube genotyping of 2 close polymorphisms
- Source :
- Clinical chemistry (Online) 54 (2008): 227–229. doi:10.1373/clinchem.2007.095703, info:cnr-pdr/source/autori:Piccioli P; Serra M; Pedemonte S; Balbi G; Loiacono F; Lastraioli S; Gargiulo L; Morabito A; Zuccaro D; Del Mastro L; Pistillo MP; Venturini M; De Angioletti M; Notaro R/titolo:Hexaprimer amplification refractory mutation system PCR for simultaneous single-tube genotyping of 2 close polymorphisms/doi:10.1373%2Fclinchem.2007.095703/rivista:Clinical chemistry (Online)/anno:2008/pagina_da:227/pagina_a:229/intervallo_pagine:227–229/volume:54
- Publication Year :
- 2008
- Publisher :
- American Association for Clinical Chemistry, Washington, DC , Stati Uniti d'America, 2008.
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Abstract
- Tetraprimer amplification refractory mutation system PCR (T-ARMS-PCR) is a simple and inexpensive genotyping method for differentiating both alleles of a polymorphism/mutation (both single-nucleotide polymorphisms and small insertions/deletions) with a single-tube PCR (1). In T-ARMS-PCR, a pair of common (outer) primers produces a non–allele-specific control amplicon and in combination with 2 allele-specific (inner) primers (designed to anneal in the opposite orientation) produces 2 allele-specific amplicons. These allele-specific amplicons have different sizes because the polymorphism/mutation is asymmetrically located with respect to the common primers. Thus, the amplicons can be separated by standard gel electrophoresis. T-ARMS-PCR has also been designed in a multiplex fashion to genotype more than one polymorphism/mutation by a single-tube PCR (2). We describe a modified multiplex T-ARMS-PCR, the hexaprimer ARMS-PCR (H-ARMS-PCR), which is for when 2 polymorphisms are close in the sequence. H-ARMS-PCR uses only 6 primers and provides direct information about haplotype structure. The CTLA4 gene (cytotoxic T-lymphocyte–associated protein 4; also known as CD152) is a negative regulator of T-cell function (3). The CTLA4 polymorphisms −318 C>T (rs5742909) and +49 A>G (rs231775) are associated with susceptibility to autoimmune diseases and cancer (3)(4). To genotype these 2 polymorphisms, which are only 365 bp apart in the 5′ region of the CTLA4 gene, we designed an H-ARMS-PCR that combines a single pair of common primers and 2 pairs of allele-specific primers in the same tube (Fig. 1A⇓ ). Figure 1. Polymorphism genotyping by H-ARMS-PCR. (A), H-ARMS-PCR for CTLA4 −318 C>T (rs5742909) and …
- Subjects :
- Gel electrophoresis
Genetics
Polymorphism, Genetic
Genotype
Biochemistry (medical)
Clinical Biochemistry
Haplotype
Reproducibility of Results
Amplicon
Biology
Molecular biology
Antigens, Differentiation
Polymerase Chain Reaction
Aromatase
Antigen
Antigens, CD
Mutation
Humans
Multiplex
CTLA-4 Antigen
Allele
Genotyping
DNA Primers
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Clinical chemistry (Online) 54 (2008): 227–229. doi:10.1373/clinchem.2007.095703, info:cnr-pdr/source/autori:Piccioli P; Serra M; Pedemonte S; Balbi G; Loiacono F; Lastraioli S; Gargiulo L; Morabito A; Zuccaro D; Del Mastro L; Pistillo MP; Venturini M; De Angioletti M; Notaro R/titolo:Hexaprimer amplification refractory mutation system PCR for simultaneous single-tube genotyping of 2 close polymorphisms/doi:10.1373%2Fclinchem.2007.095703/rivista:Clinical chemistry (Online)/anno:2008/pagina_da:227/pagina_a:229/intervallo_pagine:227–229/volume:54
- Accession number :
- edsair.doi.dedup.....b0020f8053a91c70158e93f000cb2744
- Full Text :
- https://doi.org/10.1373/clinchem.2007.095703