Mutational analysis of Sanfilippo syndrome type A (MPS IIIA): identification of 13 novel mutations

A modified version of the ammonium acetate salting out method was used to extract genomic DNA from either venous blood or fibroblast cell lines of the patients. 15 16 Each of the eight exons and intron/exon boundaries of the sulphamidase gene was amplified by PCR using intronic primers (table 1 ). 1...

Full description

Saved in:
Bibliographic Details
Published in:Journal of medical genetics 2000-09, Vol.37 (9), p.704-707
Main Authors: BEESLEY, CLARE E, YOUNG, ELISABETH P, VELLODI, ASHOK, WINCHESTER, BRYAN G
Format: Article
Language:English
Subjects:
Deoxyribonucleic acid
DNA
DNA - chemistry
DNA - genetics
DNA Mutational Analysis
Enzymes
Funding
Genes
Genotype
Genotype & phenotype
Haplotypes
Humans
Hydrolases - genetics
Letters to the Editor
Mucopolysaccharidosis III - enzymology
Mucopolysaccharidosis III - genetics
Mucopolysaccharidosis III - pathology
Mutagenesis, Insertional
Mutation
Mutation, Missense
Patients
Polymorphism, Single-Stranded Conformational
Proteins
Sequence Deletion
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A modified version of the ammonium acetate salting out method was used to extract genomic DNA from either venous blood or fibroblast cell lines of the patients. 15 16 Each of the eight exons and intron/exon boundaries of the sulphamidase gene was amplified by PCR using intronic primers (table 1 ). 1 SFA1(+) 3-19 1-150 M13 (-21)- GCGGGAGACCAGAGAGC 301 1.5 64 SFA1(-) 267-251 M13 rev-CAGCGGGGGATACAAGG BstNI (118+183) 2 SFA2(+) 11-30[dagger] M13 (-21)- ACAGTCCCAGCCTCCCTACT 362 1.5 60 SFA2(-) 336-319 M13 rev-ATGGGAGACGTGGCAGAG HaeII (142+220) 3 SFA3(+) 79-95[double dagger] M13 (-21)- GGGCCATGGGAGAACAG 306 1.5 62 SFA3(-) 348-330 M13 rev- CGTGCCTTGGTACAAGGTG ApaLI (129+177) 4 SFA4(+) 426-442[double dagger] M13 (-21)- CGAGAACCCACAGTGCG 338 1.5 64 SFA4(-) 727-711 M13 rev- GTGCTCTGGTACCGGCC BsaAI (138+200) 5 SFA5(+) 27-43§ M13 (-21)- CCTTCCTGTGCCACGTG 354 1.0 62 SFA5(-) 344-323 M13 rev- TCTTTCTTCATCATCTAGGGCC RsaI (139+215) 6 SFA6(+) 493-511§ M13 (-21)- TGTTCTAAGCCTGGCTCCC 262 1.0 62 SFA6(-) 718-701 M13 rev- CTGCCACACTGGACCCTC StyI (130+132) 7 SFA7(+) 40-56¶ M13 (-21)- GGATGCAGCAGCAGGTG 422 1.5 64 SFA7(-) 425-406 M13 rev- AGGTAATGGGTGTGGAGCAG RsaI (219+203) 8a SFA8a(+) 1286-1304¶ M13 (-21)- TTGGATTGGAGAAGGGAGC 493 1.5 66 SFA8a(-) 1742-1721 M13 rev- CCGGTAGTAGTAATGACGGAGG HaeII (230+263) 8b SFA8b(+) 1632-1651¶ M13 (-21)- CCCATCGACCAGGACTTCTA 407 1.0 62 SFA8b(-) 2002-1984 M13 rev- GGATGTGTCTGGGACATGC SacI (143+264) [dagger][double dagger]§¶Positions of primers are numbered according to Genbank database entry u60107, u60108, u60109, u60110, and u60111, respectively. 7 A typical PCR reaction using 100 ng of genomic DNA contained 25 pmol of each primer, 1 x NH4 reaction buffer (Bioline), 4% (v/v) DMSO (dimethylsulphoxide), 0.2 mmol/l dNTPs, and 0.5 [micro]l (2.5 units) BioProTM DNA polymerase (Bioline) (added after a hot start).
ISSN:0022-2593
1468-6244
1468-6244
DOI:10.1136/jmg.37.9.704