Monitoring specific nucleic acid sequences Assignment Help

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Monitoring specific nucleic acid sequences:

The rate of establishing of double-stranded hybrids depends on the concentration of the two single-stranded species. That can be used to measure the concentration of either specific RNA or DNA sequences in a complex mixture.  The first job is to prepare a single-stranded DNA probe for instance a DNA fragment which is complementary to the nucleic acid being assayed.  This should be one strand of a DNA restriction fragment, a synthetic or cloned DNA oligonucleotide.  It must be labeled in the order to be able to detect the formation of hybrids among it and the goal nucleic acid. While most labeling used to be through the incorporation of a radioisotope, nonradioactive chemical labels are now frequent used instead. For instance, a DNA probe can be labeled with digoxygenin, a steroid, through using digoxygenin-labeled dUTP during DNA synthesis. Hybrids holding the digoxygenin-labeled DNA  probe  can  then  be  detected  using  antidigoxygenin antibody   linked  to  a  fluorescent   dye.  Irrespective   of the technique of probe labeling the DNA probe is incubated with the nucleic acid sample (the 'target' DNA or RNA) and then nuclease is added to degrade any unhybridized single- stranded probe. The value of labeled probe remaining indicates the concentration of the target nucleic acid in the sample.

The hybridization   conditions for example  temperature and salt  concentration   can  be varied  so  as  to  govern  the  kind  of  hybrids  formed.  The  conditions  should  be arranged  so which only perfectly  matched  hybrids  are stable and therefore assayed conditions  known  as  high  stringency.  In the other words, the conditions may be such which even poorly complementary hybrids are stable and will be detected low stringency.  Therefore through varying the reaction conditions it is probably to detect and quantify only those goals sequences which are identical to the DNA alternatively or probe, to detect and quantify related sequences. The Hybridization of nucleic acid probes with genomic DNA, for instance, can be used to measure the copy number of particular DNA sequences in the genome.  The Hybridization  of a DNA probe with cellular RNA as the goal will indicate the concentration of the corresponding  RNA  transcript  and  therefore  provides  information  about  the  stages  of gene expression.  Different  of the methodology  even permits determination  of the transcriptional   Start  and  Stop  sites  and  the  number  and  location  of  intron sequences in protein-coding  genes.

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