Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients.

The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome.

Screening for mutations in haemophilia A patients by multiplex PCR-SSCP, Southern blotting and RNA analysis: the detection of a genetic abnormality in the factor VIII gene in 30 out of 35 patients.

The molecular characterization of mutations in haemophilia A patients in this study was carried out by PCR-SSCP, Southern blotting, and reverse transcribed-PCR. A multiplex PCR in which four to eight exons were co-amplified was developed to reduce the time needed for screening the coding region of the factor VIII gene. PCR-SSCP was used to screen for small molecular defects, and reverse transcriptase PCR combined with Southern blotting was used to screen DNA for the inversions that occur frequently in intron 22 of the factor VIII gene.

Denaturing and non-denaturing gel electrophoresis as methods for the detection of junctional diversity in rearranged T cell receptor sequences.

Two nucleic acid gel electrophoresis techniques were tested as a possible tool for analyzing junctional diversity in rearranged T cell receptor (TcR) sequences in order to define the extent of T cell heterogeneity. For this purpose denaturing gradient gel electrophoresis (DGGE) as well as non-denaturing gel electrophoresis (nDGE) techniques have been studied. Detection of junctional diversity is based on mobility shifts, caused by nucleotide sequence polymorphism, of polymerase chain reaction amplified rearranged TcR sequences.