Klug, Old Activity 12

Klug, Old Activity, DNA Sequencing from Concepts in Genetics text Klug, NEW Activity 19.4, DNA Sequencing

* In its revised format include segments 1 to 5 of the tutorial called DNA Sequencing from Concepts in Genetics. These parts should be separated from the original tutorial then combined to form this new tutorial, Activity 19.4. It will have it's own Introduction, Summary, and Post-quiz. * Remove the screen that shows on selecting “module quiz” asking if the user wishes to take the quiz.

New organization: Activity 19.4, DNA Sequencing Introduction (New 19.4.1) - This will not be a part of the Flash animation, but will be written in html. - This is an entirely new Introduction page. The first segment in the extant tutorial will become the second segment in 19.4. - Use the image from segment 2, screen 2 of the old tutorial the chart of colored peaks and the associated DNA sequences (see picture at right). Center this image in the graphics window and write the title “DNA Sequencing” over the top. - Write the following text in the text window:

“DNA Sequencing is a laboratory method of determining the nucleotide sequence of a DNA fragment. The most popular method, sometimes called dideoxysequencing, was worked out by Frederick Sanger in 1974, and so is also called Sanger sequencing. The method utilizes DNA polymerase in vitro to perform a series of interrupted syntheses along a template strand, creating fragments of different lengths.

Originally Sanger sequencing was performed with radioactively labeled dideoxynucleotides but this is rapidly giving way to the use of non-radioactive dideoxysequencing using fluorescent dyes.

This activity explores the theory of Sanger sequencing – how it works – and how the method has now been adapted so that the process can be performed by an automated sequencer.”

- This is the end of the segment. Animation (19.4.2.) Theory of Sanger Sequencing - Change the name of this segment to “Theory of Sanger Sequencing” from “Introduction to DNA Sequencing”. - This segment is a revision of the old original first segment, Introduction to DNA Sequencing. - Bring up the same starting image showing “Portion of human gene for cystic fibrosis” in 19.4.2.1. - Scroll the text of 19.4.2.1 (“DNA sequencing is a procedure…” from the bottom of the text window to the top, stopping when the first line reaches the top of the window. Pause 3 seconds and dissolve to the animation of 19.4.2.2. - Dissolve to the first frame of 19.4.2.2, but pause 3 seconds before actually starting the animation. This animation shows TTCGACC (Primer) flying in from the left. Pause 4 seconds after this concludes and move to 19.4.2.3. - Run 19.4.2.3 showing DNA polymerase fading in (do it more slowly). Pause 3 seconds and move on to 19.4.2.4. - Run 19.4.2.4 showing DNA polymerase moving to the right, creating a growing DNA strand. Pause 3 seconds after this is complete and dissolve to the new text and image of 19.4.2.5 - In 19.4.2.5, an orange/yellow/blue normal and dideoxynucleotide are displayed. - Scroll the text of 19.4.2.5 (“Dideoxynucleotide triphosphates (ddNTPs) lack…” from the bottom of the text window to the top, stopping when the top line reaches the top. Pause 2 seconds and dissolve to the next screen, 19.4.2.6. - Bring up 19.4.2.6 showing a long DNA sequence – AAGCTGGCAC…etc and 5 primers underneath reading TTCGACC. Pause 8 seconds after bringing this up and move to the animation of 19.4.2.7. - Run 19.4.2.7 showing the bottom 4 TTCGACC sequencing elongating to end in a red colored letter. Pause 4 seconds at the end of this and dissolve to the image and text of 19.4.2.8. - Bring up the image of the gel diagram showing bands in a black rectangle in 19.4.2.8 and the text (static) “When these fragments are….” the top. - This is the end of the segment. - Set scroll flags at the start of the following sequences: “Primer” at 19.4.2.2, “Elongation” at 19.4.2.4, “ddNTPs” at 19.4.2.5, “Termination” at 19.4.2.6, and “Gel” at 19.4.2.8. Animation (New 19.4.3) Manual Sequencing - Bring up the full screen showing seven sequences, the lower 6 getting progressively longer as you move downwards (plus caption). Pause 8 seconds after this comes up and dissolve to the new image and text of 19.4.3.2. - Bring up the photo of the sequencing gel in 19.4.3.2 and the text “Each of these reactions is…” Hold for 5 seconds and dissolve to the next image and text. - In 19.4.3.3, bring up the white rectangle image of the gel with Large DNA and Small DNA and the text “The smallest fragments of DNA….” Pause 5 seconds and dissolve to the animation of 19.4.3.4. - Run the animation of 19.4.3.4 which shows the Large and Small DNA bands moving downwards in the gel. Pause 3 seconds and dissolve to the next text and image. - In 19.4.3.5, a gray shadow box comes up over the extant gel image and the text “After the sequencing gel has run….” comes up. Pause 3 seconds and dissolve to the next image and text of 19.4.3.6. - Bring up the photo of the X-ray gel showing bands under the columns GATC in 19.4.3.6. - Scroll the text of 19.4.3.6 from the bottom to the top of the text window, stopping when the top line reaches the top - This is the end of the segment. - Set Scroll flags at: “Gel box” at 19.4.3.2, “Migration” at 19.4.3.3, “Autorad” at 19.4.3.5, and “Results” at 19.4.3.6. Animated (New 19.4.4) Automated Sequencing - Bring up the full screen image showing a long DNA sequence followed by 7 progressively longer ones, and the caption “When using an automated sequencer…” Pause 7 seconds and dissolve to the next screen. - In 19.4.4.2, bring up the diagram showing two rows of colored peaks along a chain of nucleotides, and the caption “The result is…” - This is the end of the segment. - Set one scroll flag called “Electropherogram” at 19.4.4.2 Summary (New 19.4.5) -This will not be programmed as part of the Flash animation. - Convert this page to our new format. Post-quiz (New 19.4.6) -This will not be programmed as part of the Flash animation. - Delete screen asking if user wants to take the quiz.

First Pass Comments from Eric Stavney 10/22/03

1. On the introductory page, it would be nice to lose the black frame around the graphic or at least tighten it up so that it runs along the actual edges of the image on the top and bottom. [Andy 10/23: Please make these changes] 2. The third sentence of the text for the Introductory page needs to be changed so the phrase “in vitro” is in italics: “….utilizes DNA polymerase in vitro…..” [Andy 10/23: Please make these changes] 3. The text scrolling speed in 19.4.2 is way too slow. Please speed it up so maybe 1- 2 lines clear the bottom per second. Check on our standard speed. [Andy 10/23: Please make these changes] 4. At the Elongation scroll flag of 19.4.2, several labels and objects are added in a sequence in the animation. Please make these labels and the egg-shaped polymerase fade in rather than pop in abruptly. [Andy 10/23: The hard fade is acceptable.] 5. The label on the DNA polymerase in 19.4.2.3 is of color that I can barely read – off-white. Please chose a different color, like the standard gold to emphasize these words. [Andy 10/23: Please make this change AND move the “Growing DNA strand” label and arrow so that its tip is not inside the egg.] 6. The transition across the Gel scroll flag in 19.4.2 needs to be a dissolve – fade out and fade in --- instead of an abrupt cut. [Andy 10/23: The hard fade is acceptable.] 7. The transition at the Gel Box scroll flag of 19.4.3 needs to be a dissolve – fade out and fade in --- instead of an abrupt cut. [Andy 10/23: The hard fade is acceptable.] 8. The transition at the Migration scroll flag of 19.4.3 needs to be a dissolve – fade out and fade in --- instead of an abrupt cut. [Andy 10/23: The hard fade is acceptable.] 9. The transition at the Autorad scroll flag of 19.4.3 needs to be a dissolve – fade out and fade in --- instead of an abrupt cut. [Andy 10/23: Please use a SOFT fade here.] 10. Please change the Autorad scroll flag of 19.4.3 to say “Autoradiography” [Andy 10/23: Make this change.] 11. The transition at the Results scroll flag of 19.4.3 needs to be a dissolve – fade out and fade in --- instead of an abrupt cut. [Andy 10/23: The hard fade is acceptable.] 12. Where did the text come from that follows the unit in 19.4.3.6? I’ve never seen it before. It reads “Open a new browser window (Netscape or Explorer) and log into….” I don’t know how this got here, but it doesn’t make sense to have it here. Please delete it and end the segment at the end of the first unit of text in 19.4.3.6. If somebody insists on keeping it, at least fade out the first text before bringing up this new unit. [Andy 10/23: Delete this material.] 13. The transition at the Electropherogram scroll flag of 19.4.4 needs to be a dissolve – fade out and fade in --- instead of an abrupt cut. [Andy 10/23: Please use a SOFT fade here.] 14. Woops --- it looks like this whole tutorial isn’t scalable. I was finding the graphic 19.4.4.2 too small to read and tried to enlarge it with the browser window but no go. Make the segments scalable as in all our other tutorials. I’d also suggest moving the text of 19.4.4.2 to the right side, enabling you to enlarge the graphic of the colored peaks. [Andy 10/23: Please make this universal change. ALL animations should be dynamically scale when the size is resized.] Second Pass Comments from Eric Stavney 10/20/03

1. Looks good to me. [Andy 10/31: The color of the screen title should be the brown used in the other tutorials and not the black used here.] [Andy 10/31: Otherwise, looks great.]