Conformation sensitive gel electrophoresis for detection of factor X gene mutations

Abstract Our previous findings suggested that coagulation factor XFriuli could be functionally defective owing to a point mutation in the portion of the factor X gene coding for the fully activated heavy chain. To verify the existence of this postulated change, we analyzed all eight exons of the normal and Friuli factor X gene. Each exon was amplified from genomic DNA using the polymerase chain reaction and cloned into the plasmid pUC19. The amplified DNA inserts were subjected to direct sequencing by the dideoxy chain termination method with forward and reverse oligonucleotide sequencing primers. A point mutation (C to T transition at nucleotide position 19,297) that results in coding for serine (TCC) in place of proline (CCC) at amino acid position 343 was found. This substitution involves a highly conserved proline residue oriented spatially close to both the cleavage site of the zymogen and the active site of the enzyme and explains the previous observations of discrete biochemical and functional differences between factor XFriuli and normal factor X. The mutation abolished an HgiCI restriction site present in the normal factor X gene, and this change constitutes the basis for a convenient method for screening individuals carrying this molecular defect. Proline343 is in conserved region 5 of the serine protease superfamily to which factor X belongs and is part of a 14- residue L*****P******C motif that occurs in at least 16 other enzymes. Computer analysis suggests that the motif may be an essential aspect of conformational features important to functional properties of factor X as well as other serine proteases.

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Rapid Detection of β-Globin Gene Mutations and Polymorphisms by Temporal Temperature Gradient Gel Electrophoresis

Clinical Chemistry ◽

10.1373/49.5.777 ◽

2003 ◽

Vol 49 (5) ◽

pp. 777-781 ◽

Cited By ~ 16

Author(s):

Ramachandran V Shaji ◽

Eunice Sindhuvi Edison ◽

Balasubramanian Poonkuzhali ◽

Alok Srivastava ◽

Mammen Chandy

Keyword(s):

Temperature Gradient ◽

Gel Electrophoresis ◽

High Throughput Screening ◽

Globin Gene ◽

Mutation Screening ◽

Gene Mutations ◽

Cost Effective ◽

Mendelian Disease ◽

Gradient Gel Electrophoresis ◽

Temperature Gradient Gel Electrophoresis

Abstract Background: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. Methods: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 β-thalassemia genes from homozygous β-thalassemia patients and unrelated individuals with heterozygous β-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE. Results: We found common mutations in 753 β-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the β-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning β-globin gene were suspected. Conclusions: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.

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