Course: SAT 170 Introductory Biotechnology

Unit 7 Reading and Multimedia Assignment

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- Unit 7 Key Terms (Selected)

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“Library screening for clones of interest” by Boundless, CC BY SA 3.0

Link: https://www.boundless.com/biology/biotechnology/dna-and-biotechnology/library-screening-for-clones-interest/

Reading Assignment: Library screening for clones of interest
To find the gene of interest among clones, different methods can be used, including nucleic acid hybridization, PCR, and DNA sequencing.

Southern

The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library. Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously-cloned DNA fragment into a different plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained. This may be accomplished through a very wide range of experimental methods, including the use of nucleic acid hybridizations, antibody probes, polymerase chain reaction, restriction fragment analysis, and/or DNA sequencing.

In molecular biology, a hybridization probe, or nucleic acid probe, is a fragment of DNA or RNA of variable length (usually 100-1,000 bases long) that is used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a nitrocellulose or nylon membrane, or in situ (see above figure).

To detect hybridization of the probe to its target sequence, the probe is tagged (or labeled) with a molecular marker of either radioactive or (more recently) fluorescent molecules; commonly used markers are 32P (a radioactive isotope of phosphorus incorporated into the phosphodiester bond in the probe DNA) or Digoxigenin, which is a non-radioactive antibody-based marker. DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Normally, either X-ray pictures are taken of the filter, or the filter is placed under UV light. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied. High stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile cDNA target is hybridized.

Once a clone containing the gene of interest is identified, that clone can be multiplied exponentially by growing it in a liquid medium. This produces millions of copies of the gene, carried in millions of cells.