{"id":393,"date":"2019-07-10T10:01:10","date_gmt":"2019-07-10T07:01:10","guid":{"rendered":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/?page_id=393"},"modified":"2019-09-08T13:29:16","modified_gmt":"2019-09-08T10:29:16","slug":"reading-the-results-page","status":"publish","type":"page","link":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/home\/exploring-the-genotype-phenotype-relations-in-cystic-fibrosis\/task-no-2-detection-of-a-cftr-gene-mutation-using-pcr-and-the-tool-primer3plus\/designing-pcr-primers-for-the-diagnosis-of-cftr-gene-mutation-using-the-tool-primer3plus\/reading-the-results-page\/","title":{"rendered":"Reading the results page"},"content":{"rendered":"

Lets go back to the main page (“Main” tab).
\nAs an input, the tool receives a DNA sequence that include the target DNA for amplification. Of the normal CFTR coding sequence (CDS), the researchers chose a segment of 450 nucleotides around the F508del mutation site (located in positions 201-203 in this DNA segment). Lets copy and paste the desired sequence from the normal CFTR gene to the designated window (copy the sequence only, without the title line). Click “Pick Primers” in order to design primer pairs using the tool. The results obtained are displayed in screens 4-6. The tool presents 5 possible primer pairs that meet the default settings we reviewed before, and indicates the characteristics of the primers and the expected amplification product for each pair. For the first primer pair, we can see the sequence of each primer (in the field “Sequence”), the position in the template (input) sequence in which the primer starts (“Start”), its length (“Length”), its annealing temperature (“Tm”) and more details. The length of the expected amplification product from using these primers is also indicated (“Product Size”, Screen 4). Following this, we can see the graphic description of the primers and their location on the template sequence.<\/p>\n

\"Screen<\/figure>\n

Screen 4: The results page – the characteristics of the first primer pair, the expected amplification product and its location on the template<\/strong><\/p>\n

Later, the same data on the primers and the expected amplification product is detailed for the other primer pairs as well (Screen 5).<\/p>\n

\"Screen<\/figure>\n

Screen 5: The results page – the characteristics of the rest of the primer pairs and the expected amplification products<\/strong><\/p>\n

Lets look at the 5 primer pairs designed by Primer3Plus.<\/p>\n

10. What can we say about the designed primer pairs?”><\/p>\n

    \n
  1. All the primers are 20 nucleotides long with an annealing temperature of 58-60\u00b0C.<\/li>\n
  2. All the primers are 20 nucleotides long with an annealing temperature of 45-50\u00b0C<\/li>\n
  3. All the primers are 20 nucleotides long with an annealing temperature of 45-55\u00b0C.<\/li>\n
  4. It is only incidental that the primer pairs have similar characteristics.<\/li>\n<\/ol>\n

    The answer is:<\/strong>\u00a0A. All the primers are 20 nucleotides long with an annealing temperature of 58-60\u00b0C, which meets the default primer settings (as mentioned in the tab “General Settings”).<\/p>\n

    11. What can we say about the expected amplification products?<\/p>\n

      \n
    1. All the expected amplification products are 165 bp long (bp = base pairs).<\/li>\n
    2. All the expected amplification products share the sequence in positions 20 to 170.<\/li>\n
    3. All the expected amplification products are 20 bp long.<\/li>\n
    4. All the expected amplification products share the sequence in positions 20-230.<\/li>\n<\/ol>\n

      The answer is:<\/strong>\u00a0B. For all the five designed primer pairs, the left primer starts prior to position 20 and the right primer starts at position 179 or downstream. Keeping in mind that the primers define the ends of the expected amplified segment in the PCR, positions 20 to 170 are necessarily inside the amplified region. Of note, statement A is incorrect since the amplification product from the fifth primer pair is 235 bp long; C is wrong since the 20 bp length mentioned refers to the length of the primers rather than the products; D is true only for the fifth primer pair but not for primer pairs 1-4.<\/p>\n

      12. The F508del deletion mutation is located in positions 201-203 in the 450 nucleotides long DNA sequence the researchers focus on. In the sequence from the healthy subject the sequence CTT will appear in these positions, while in the mutant allele the nucleotides CTT are missing, and in their place TGG from positions 204-206 in the normal sequence appear. Will any of the designed primers amplify the segment that includes the mutation site?<\/p>\n

      Teachers:\u00a0<\/strong>The fifth primers pair is expected to amplify the segment from position 15 (the beginning of the left primer) to position 249 (the beginning of the right primer) and thus the product should include the F508del mutation site. The rest of the pairs will amplify segments that ends are in position 179 – a segment that will miss the relevant mutation site.<\/span><\/p>\n

      In fact, among the designed primer pairs, the fifth pair should amplify the segment that includes the F508del mutation site (Screen 6). Nevertheless, it is not certain that the primers designed by the tool will include the desired segment unless it is specifically defined to the tool.<\/p>\n

      \"Screen<\/figure>\n

      Screen 6: An optional primer pair (the fifth pair) for the amplification of a segment that includes the F508del mutation site<\/strong><\/p>\n","protected":false},"excerpt":{"rendered":"

      Lets go back to the main page (“Main” tab).
      \nAs an input, the tool receives a DNA sequence that include the target DNA for amplification. Of the normal CFTR coding sequence (CDS), the researchers chose a segment of 450 nucleotides around the F508del mutation site (located in positions 201-203 in this DNA segment). Lets copy and paste the desired sequence from the normal CFTR gene to the designated window (copy the sequence only, without the title line). Click “Pick Primers” …<\/p>\n","protected":false},"author":30,"featured_media":0,"parent":373,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template_leftnav.php","meta":{"om_disable_all_campaigns":false,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"class_list":["post-393","page","type-page","status-publish","hentry"],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/pages\/393"}],"collection":[{"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/users\/30"}],"replies":[{"embeddable":true,"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/comments?post=393"}],"version-history":[{"count":2,"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/pages\/393\/revisions"}],"predecessor-version":[{"id":398,"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/pages\/393\/revisions\/398"}],"up":[{"embeddable":true,"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/pages\/373"}],"wp:attachment":[{"href":"https:\/\/stwww1.weizmann.ac.il\/bioinfo\/wp-json\/wp\/v2\/media?parent=393"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}