A Guide to Methods in the Biomedical Sciences by Ronald B. Corley PDF

By Ronald B. Corley

Millions of tools were constructed within the quite a few biomedical disciplines, and people coated during this e-book characterize the elemental, crucial and most generally used tools in numerous various disciplines.

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Dideoxy or chain-termination sequencing (also known as Sanger sequencing) This is an enzymatic method (16) that relies on the fact that the incorporation of dideoxynucleotides (ddNTPs) rather than deoxynucleotides (dNTPs) results in the termination of elongation of DNA catalyzed by DNA polymerase. DNA polymerases works by elongation of a DNA strand complementary to a template DNA, and requires a hydroxyl (OH) group at the position for DNA polymerase to add the complementary nucleotides. Since dideoxynucleotides lack a OH group present in deoxynucleotides, the incorporation of a ddNTP terminates elongation.

Is added in limiting concentrations. Included in each reaction is a radioactive nucleotide (usually is used). The DNA polymerase initiates chain elongation in a direction, starting at end of the oligonucleotide primer that has been annealed to the DNA template. The DNA polymerase adds deoxynucleotides, which are selected by base-pair matching, to the elongating DNA chain. DNA polymerases can incorporate both dNTPs and ddNTPs as substrates. As a result, chain elongation is terminated whenever a ddNTP is incorporated since they have no hydroxyl groups.

This system can be used to identify interacting proteins from a variety of eukaryotic species that mediate a variety of functions, from signal transduction to transcription, and has been one of the most useful systems developed to identify interacting partners. Fluorescence resonance energy transfer (FRET) Fret is another powerful method for identifying proteins that interact within live cells. FRET depends on the ability to stably or transiently express each of two different interacting proteins in a cell that are labeled with fluorescent molecules that are capable of transferring energy.

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