21-10-2014_16-13-52 / рис.2-лекц-9

Screening

Once a particular DNA fragment is identified, it can be isolated and amplified to determine its sequence.  If we know the partial sequence of a gene and want to determine its entire sequence, the probe should contain the known sequence so that the detected DNA fragment may contain the gene of interest.  

Figure 9-B-3.  Screening of a specific DNA fragment.  After recombinant  virions form plaques on the lawn of E. coli, the nitrocellulose filter (membrane) is placed on the surface of the petri dish to pick up  phages from each plaque.  Then, the filter is incubated in an alkaline solution to disrupt the virions and release the encapsulated DNA, which is subsequently denatured.  Next, the probe is added to hybridize with the target DNA fragment, whose position may be displayed by autoradiography.