Molecular cloning, expression and characterization of mouse single-chain fragment variable antibody against citrus tristeza virus

The sequences encoding the mouse heavy-chain [V[H]] and light-chain [V[L]] variable region genes were isolated by PCR and joined into a single-chain Fv [scFv] DNA by using a DNA linker encoding [Gly4Ser][3] peptide. The scFv DNA fragment was cloned into the phagemid pCANTAB5E and expressed in Escherichia coli as a fusion protein with M13 phage p3 polypeptide and E tag. The scFv fusion protein was displayed on the surfaces of recombinant M13 phages in the presence of the helper phage M13K07. High-affinity scFv phage-bodies against citrus tristeza virus [CTV] were enriched through affinity selection on immobilized recombinant CTV coat protein preparations. The selected recombinant phages were used to infect Escherichia coli HB2151 for the production of soluble scFv antibodies. One selected clone in HB2151 secreted a soluble scFv antibody that detected CTV in extracts of infected citrus plants with a sensitivity comparable to that of a commercial monoclonal antibody. The nucleotide sequence of the light-chain and heavy-chain portions were closely related to other published scFv against CTV. The potential of this scFv was demonstrated in routine field testing using an inexpensive tissue print-ELISA

Rapid carrier screening using short tandem repeats in the phenylalanine hydroxylase gene

Phenylketonuria [PKU] is an autosomal recessive genetic disorder caused by defects in the phenylalanine hydroxylase [PAH] system. Our work aimed to screen the PAH locus for the presence of potentially useful short tandem repeats [STR] as markers for carrier detection in PKU families in Egypt, and to determine the level of PAH heterozygosity within the Egyptian population. The system contains at least eight independent alleles in the Egyptian population, transmitted in a Mendelian fashion. Variations in the number of STR in the 16 families studied gave rise to polymorphisms that proved to be suitable markers for PKU carrier detection and prenatal diagnosis. The most frequent allelic fragment size in PKU patients was 246 bp [35.7%], which together with a fragment of 254 bp accounted for 60.7% of the mutant chromosomes

Molecular diagnosis of spinal muscular atrophy in Egyptians

This study was carried out with 33 spinal muscular atrophy [SMA] patients. DNA molecular studies of the SMA gene on the long arm of chromosome 5 [5q11.2q13.3] revealed homozygous deletion of exon 7 in 55% of cases, 36% of whom also had a homozygous delition of exon 8. The adult patients were heterozygous for an abnormal size exon 8. The remaining patients had either compound heterozygote deletion of exons 7 and 8 or were normal for both. There may therefore be 5q-unlinked SMA or SMA due to other mutations. Detection of deletions of SMA exons 7 and 8 is a powerful diagnostic test in patients with SMA, but other mutations among Egyptians must also be sought