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- Paunio, T, et al.
(författare)
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Preimplantation diagnosis by whole-genome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA
- 1996
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 42:9, s. 1382-1390
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a new method for preimplantation diagnosis of inherited diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer random primers (primer extension preamplification, PEP) with a single locus-specific PCR amplification, followed by detection of the mutation by solid-phase minisequencing. The procedure was evaluated by detecting three disease-causing mutations and seven polymorphic nucleotides located on different human chromosomes from single granuloma and blastomere cells. The correct genotype of the cell was identified at 96% of the nucleotide positions analyzed, showing that a representative part of the genome is amplified during PEP. We estimate that PEP yielded at least 1000 copies of the genome. The quantitative nature of the solid-phase minisequencing method allowed us to notice that preferential amplification of one allele occurs at heterozygous loci during PEP, which is a potential problem in preimplantation diagnosis.
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| 3. |
- Paunio, T, et al.
(författare)
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Tissue distribution and levels of gelsolin mRNA in normal individuals and patients with gelsolin-related amyloidosis.
- 1997
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 406:1-2, s. 49-55
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Tidskriftsartikel (refereegranskat)abstract
- We measured quantitatively the mRNA levels of intracellular and secretory forms of gelsolin, an actin-modulating protein, in human tissues from subjects of different ages. The intracellular gelsolin mRNA constituted the major type of gelsolin steady-state mRNA in all tissues analyzed. Both forms of gelsolin were expressed in most adult tissues, with particularly high mRNA levels in all types of muscle and interestingly in skin. Between the adult and infantile tissues the most striking difference in expression levels was found in liver, as the adult liver contained only a subtle amount of gelsolin mRNA. Skin and muscle samples from patients with gelsolin-related amyloidosis (FAF), with significantly increased concentrations of serum gelsolin, did not reveal an increased expression of the gene, and both mutant and wild-type alleles were expressed in equal amounts. The high level of expression of the gelsolin gene in the skin in general could locally contribute to the characteristic skin amyloidosis in FAF patients.
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| 4. |
- Syvänen, Ann-Christine, et al.
(författare)
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DNA diagnosis and identification of carriers of infantile and juvenile neuronal ceroid lipofuscinoses
- 1997
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Ingår i: Neuropediatrics. - : Georg Thieme Verlag KG. - 0174-304X .- 1439-1899. ; 28:1, s. 63-66
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Tidskriftsartikel (refereegranskat)abstract
- The recent identification of the genes and the mutations underlying infantile neuronal ceroid lipofuscinosis and juvenile onset neuronal ceroid lipofuscinosis facilitates specific DNA-based diagnostics for the disorders. We have developed a solid-phase minisequencing test for the identification of the major Finnish INCL mutation, an A to T transversion at nucleotide position 364 of the palmitoyl protein thioesterase gene on chromosome 1. This test has been applied for prenatal diagnosis and for identification of disease carriers in INCL families. For population-based screening for INCL carriers the coverage of the test would be 98%. In addition, by combining the solid-phase minisequencing test with whole genome preamplification, we have developed a procedure that allows reliable identification of the INCLFin-mutation in single blastomeres from in-vitro-fertilized embryos. This method is applicable for preimplantation diagnosis, and thus it offers an alternative to early prenatal diagnosis in the prevention of INCL. A modification of the solid-phase minisequencing test was devised for detection of the major INCL mutation, a 1.02 kb deletion in the CLN3 gene on chromosome 16. The coverage of this test for diagnosis of INCL and identification of carriers is 90% in Finland and > 80% worldwide.
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