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Genotyping of the r...
Genotyping of the reduced folate carrier-1 c.80G>A polymorphism by pyrosequencing technology : importance of PCR and pre-PCR optimization
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- Nilsson, Torbjörn K. (författare)
- Örebro universitet,Hälsoakademin
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Löf-Öhlin, Z. M. (författare)
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Böttiger, A. K. (författare)
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(creator_code:org_t)
- 2009-07-08
- 2008
- Engelska.
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Ingår i: Scandinavian Journal of Clinical and Laboratory Investigation. - Oslo : Taylor & Francis. - 0036-5513 .- 1502-7686. ; 68:2, s. 166-170
- Relaterad länk:
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https://urn.kb.se/re...
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visa fler...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- When developing a genotyping assay by Pyrosequencing™ technology for the RFC1 (SLC19A1) c.80G>A polymorphism (rs1051266), unequal peak heights in the pyrograms were observed, probably due to unequal amplification of the mutated and wild-type alleles. This rarely occurring problem could potentially render assignment of heterozygous genotypes uncertain. When the PCR conditions were studied, it was found that substitution of the dGTP nucleotide in the master mix by dGTP and dITP in proportion 1:1 largely overcame this problem. Heat denaturation of the DNA at 95°C before PCR also counteracted the problem. A combination of these two modifications of the standard pyrosequencing PCR protocol gave the best results. We conclude that, with these modifications, the RFC1 c.80G>A SNP can be reliably assayed by pyrosequencing.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Medicinsk genetik (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Medical Genetics (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Farmakologi och toxikologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Pharmacology and Toxicology (hsv//eng)
Nyckelord
- Aged
- Base Sequence
- DNA Primers
- Female
- Genotype
- Humans
- Male
- Membrane Transport Proteins/*genetics
- Nucleic Acid Denaturation
- Polymerase Chain Reaction/*methods
- Polymorphism; Genetic
- MEDICINE
- MEDICIN
- Clinical genetics
- Klinisk genetik
- Pharmacology
- Farmakologi
- Medicine
- Medicin
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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