| 1. |
- Gyllensten, Ulf, et al.
(författare)
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PCR-based HLA class II typing
- 1991
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Ingår i: PCR methods and applications. - : Cold Spring Harbor Laboratory. - 1054-9803. ; 1:2, s. 91-8
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Ikonen, E, et al.
(författare)
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Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing
- 1992
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Ingår i: PCR methods and applications. - : Cold Spring Harbor Laboratory. - 1054-9803. ; 1:4, s. 234-240
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Tidskriftsartikel (refereegranskat)abstract
- We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.
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| 3. |
- Pacek, P, et al.
(författare)
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Determination of allele frequencies at loci with length polymorphism by quantitative analysis of DNA amplified from pooled samples
- 1993
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Ingår i: PCR methods and applications. - : Cold Spring Harbor Laboratory. - 1054-9803. ; 2:4, s. 313-317
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Tidskriftsartikel (refereegranskat)abstract
- We present a new method that allows rapid determination of allele frequencies at loci exhibiting length polymorphism. In this method a fluorescence-labeled PCR primer is used to amplify the polymorphic region from pooled DNA samples originating from a large number of individuals. The fluorescent PCR products are separated by gel electrophoresis on an automatic DNA sequencer and the relative amount of the PCR products are determined. The distribution of the PCR products obtained from the alleles present in the pooled samples directly corresponds to the allele frequency in the population in question. The allele frequencies at a short tandem repeat locus in the von Willebrand factor gene and at the D1S80 locus were determined in the Finnish population. We found that the allele frequencies determined by quantitative analysis of PCR products from pooled DNA samples and by analyzing individual samples were in good agreement.
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