By Elizabeth Ness MacBean Ross, James Ness MacBean, Ross
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Extra resources for A Lady Doctor in Bakhtiari Land
Detection: • Fab fragments • Anti-digoxigenin, IgG (mono or polyclonal) ❑ Advantages • Molecule of plant origin • It is conjugated to dUTP or UTP in the forms of digoxigenin X-UTP and digoxigenin X-dUTP. • Specific detection in animal tissue 14 ➫ A molecule which can be conjugated to: • An enzyme (usually peroxidase) • Colloidal gold ➫ No endogenous digoxigenin in animal tissues ➫ X represents the carbon chain, comprising between 6 and 21 atoms. ➫ The digoxigenin is attached to carbon 5 of uracil via the attached carbon chain by an enzymatic reaction.
Synthesis of labeled transcripts (amplification 10 X with labeled molecules) ➫ Possibility of using several labeled nucleotides ➫ Copies the whole insert ➫ Easily controlled ❑ Disadvantages • Insertion of the cDNA into a vector to obtain transcription • Lability of the probes • Restriction enzymes are used to linearize the plasmid. • Probes are ligated to obtain smaller fragments. ➫ Construction of this is very important. ➫ Avoid all risk of contamination by RNases. ➫ Restriction enzymes have a limited lifespan and are very labile.
Detection • Avidin or multiple avidins • Streptavidin • Anti-biotin IgG (mono or polyclonal) ❑ Advantages • The biotinylated nucleotides exist in the forms of biotin X-dUTP and biotin X-UTP. ➫ These molecules can be conjugated to: • An enzyme (usually peroxidase) • Colloidal gold ➫ The length of the chain means that the antigenic group is exposed at the edge of the hybrid. ). 2 ➫ Very short and very long carbon chains limit hybridization. 1). Treatment with proteases and checking the absence of labeling from endogenous biotins.