Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency.

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.

Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.

Molecular cloning of a functional cis-acting, Bam HI-flanked, 1.6 Kb 'Mob' cassette for demonstrating rapid conversion of col EI origin-DNA cloning vectors into conjugal form.

A 1.6 Kb mobilization (Mob) fragment originating from broad host range IncP plasmid RP4 is effectively cloned into two different Col EI-origin based cloning vectors, pBluescript II SK+ and pT-Adv, to generate pPAR-I and pPAR-II, respectively. The vectors have different genetic markers and were demonstrated to get mobilized at significant frequency into a laboratory and an enteroroxigenic strain of Escherichia coli with all the genetic markers of the recombinant clones expressing efficiently in the recipient host cells. Important restriction endonuclease recognition sequences in the multiple cloning sites of the conjugal vector DNA molecules remained unique. Significance and relevance of the current study with regard to other gene delivery system in gram negative bacteria are discussed.

A method for the simulation of normal, carrier and affected controls for PCR-RFLP screening of a genetic disease in dairy cattle.

A technique is described that may be used to create in vitro mutations in PCR templates to generate affected and carrier controls for diagnostic testing when DNA from such individuals is not easily obtained. The method is demonstrated for a PCR-RFLP diagnostic test of the genetic disorder BLAD (Bovine Leukocyte Adhesion Deficiency).

Urinary fructose excretion in health after fructose test.

Urinary excretion of fructose during three hours following the ingestion of 100 g of hydrolysed sucrose (in 200 ml) was studied in 85 normal men. This solution was better tolerated than a solution of 50 g of pure fructose and gave higher urinary excretion of fructose than with 100 g of unhydrolysed sucrose. The mean fructose excretion was 54.0 mg (S.D. 40.6 MG). The majority of subjects (63) excreted between 1 and 75 mg and the highest value was 187mg. This suggests a relatively inexpensive method for assessing the existence of porta-systemic shunts beyond normal.