Cyber-Cloning of a Human Clotting Factor IX Gene Fragment.

A component of the Lycoming College Molecular Biology and Bioinformatics Project
Jeffrey D. Newman, Lycoming College © 1999

A. Obtain Factor IX Sequences from the GenBank Database.

1. Make a folder called factor9 on your disk.

2. Open the DNASTAR program GeneQuest. On the Entrez search menu, select new text search.

3. In the New Term box, enter "coagulation factor IX". A list of sequences should appear.

4. Put check marks to the left of the complete human gene sequence (ID=182612) and the complete human mRNA sequence (ID=180552), the 3’ end of the mouse mRNA sequence (ID=193317)

5. On the entrez search menu, click batch save, then click the pulldown menu next to the word save and select checked. Click the set location button and select your factor9 folder. Click OK.

6. Open the DNASTAR program EditSeq.

7. On the file menu, choose open, then open each of the files in your factor9 folder.

8. Examine the text in the lower half of each editseq window to identify each sequence. On the file menu, choose save as and save each sequence into your factor9 folder with informative names such as "human factor IX cDNA", "human factor IX gene", and "mouse factor IX cDNA."

B. Examination of human Factor IX intron-exon organization.

1. Open the DNASTAR program MapDraw, On the File menu, choose New, then double click on the human factor IX gene sequence.

2. To obtain an uncluttered view of the sequence, it may be necessary to remove some of the features shown by altering the settings under the options menu. The goal of this step is to have the sequence displayed as shown to the right.

• Put a checkmark next to 3 letter amino acid code if there is not already one there.
• go to Enzyme Display, check none
• go to Reading Frames, check none
• go to Line Layout, make sure only DNA Ruler at the top and Space at the bottom are checked, put a check mark in the save as default box. Click OK.
• Go to bases per line, select round within page, put a check mark in the save as default box. Click OK.

3. On the features menu, select show feature list. Turn off all of the checkmarks on the right hand side by holding the left mouse button down and moving the pointer over the checkmarks.

4. Browse through the feature list, how many exons and introns does the gene have?

5. Double click on the F9-CDS (coding sequence) feature. Put a check mark in the translate on site and sequence view box, as well as all of the boxes below show on. Click on OK, then close the feature list box by clicking the X box in the upper right hand corner.

6. Scroll down through the sequence, taking particular note of the segments showing the amino acid sequence below the DNA sequence. In the example at right, what does the sequence between the two translated segments correspond to?

7. On the File menu, select save map, then save file to your factor IX folder.

8. On the enzymes menu, select new filter, then manual pick. In the name box, enter none, then click apply and OK.

9. On the Map menu, select worksheet. You may need to use the magnifying glass tool to shrink the view so that it fits within your window. The boxes in the lower part of the window correspond to the exons. Note the amount of the total sequence devoted to these exons. Are you surprised? What might be the significance of all this non-coding DNA?

10. Under the file menu, choose save map.

C. Identification of fragment to be amplified.

1. On the map menu, select site and sequence.

2. On the file menu, select find. Enter the sequence "TTGATGCATTCTGTGGAGG", then click find next.

3. Once the sequence is highlighted, go to the features menu and select new feature. Enter "FIX exon 7 primer" into the Title box. Click in the boxes on the right hand side under "Show On" to insert check marks. Click the description box, choose "primer_bind". Click the style box, choose the solid arrow as seen to the right. Click OK.

4. Repeat steps 2 and 3 using the sequence for the FIX exon 8 primer, which is "GGACCCCATGTTACTGAAG". Why is this sequence different from the sequence indicated in your lab manual? How is this sequence related to that one? Why couldn’t we use the sequence as given in the lab manual?

5. Click on the location box, then double click on the numbers in the segment box to call up the box shown at the right. Click on the opposite facing arrows button to specify that this primer corresponds to the lower strand. Click the OK button on that dialog box and the next one.

6. On the map menu, choose worksheet, then use the magnifying glass tool to zoom in on the area of exons 7 and 8 until you obtain a view as shown below.

7. Click on the exon 7 primer to highlight it. On the map menu, choose site and sequence.

8. Scroll down the sequence to the FIX exon 8 primer, hold down the shift key while you click on the arrow. The sequence between the two primers should now be selected. On the edit menu, select copy. Under the file menu, choose save map

9. Open the DNASTAR program EditSeq. On the edit menu, choose paste. On the file menu, choose save. Save the file in your factor9 folder as "PCR product".

D. Alignment of Factor IX DNA Sequences.

1. Open the DNASTAR program MegAlign.

2. On the File menu, choose enter sequences. Go to your factor9 folder and select the mouse cDNA, human cDNA, human gene and PCR product sequences by highlighting the file and clicking the "Add" button, then click done.

3. On the Align menu, select method parameters and change the Gap Penalty to 20. Then, under "evaluate", click the now button. Because the gene sequence is so large (over 38,000 bases), the alignment may take a few minutes, depending upon the speed of your computer. Consider how long this would have taken on the PCs common 5 or 10 years ago!

4. When the aligned sequences are shown on the screen, move the names in the left column around so that the gene is on top, followed by the PCR product, the human cDNA and finally the mouse cDNA. Move the solid black bar at the bottom of the alignment window to the left so that one sequence alignment window is larger than the other as shown below.

5. On the file menu, choose save, then give the file a name and save it in your factor9 folder.

6. Browse through the aligned sequences to identify the primer sequences at the ends of the PCR product. Note that these sequences are nearly identical in humans and mice. Do you think these primers would work with mouse DNA?

7. Continue browsing through the sequence alignments to identify all of the introns and exons. Do you notice any sequences that are common at the junctions? Near the 3’ end of the cDNA sequences, look for the sequence AATAAA. This sequence serves as a poly-adenylation signal that is recognized by the enzymes that cut the primary transcript at the 3’ end which then has a Poly-A tail attached.

E. Comparison of Factor IX Protein Sequences

1. Open the DNASTAR program EditSeq.

2. On the file menu, choose open, then select the human cDNA sequence from your factor9 folder. Browse through the annotation in the lower panel of the window to determine what part of the sequence codes for protein (gene or CDS).

3. Click and drag to select the coding part of the cDNA sequence (note the first and the last codons – what are they?), then on the goodies menu, select translate DNA.

4. Browse through the information in the lower panel of the window. On the file menu, choose save, name the file "human factor IX" and save it in your factor9 folder.

5. Repeat these procedures with the mouse cDNA.

6. Compare the properties of the human and mouse proteins as described in the lower panel of each amino acid sequence, then close the EditSeq program.

7. Open the DNASTAR program MegAlign.

8. On the File menu, choose enter sequences. On the bottom of the subsequent dialog box, change the "Files of type" option to "Protein Files". Then go to your factor9 folder and select the mouse and human factor IX proteins by highlighting the files and clicking the "Add" button, then click done.

9. On the Align menu, select by Clustal method,

10. After the alignment is complete, review the sequences as with the DNA sequence alignment. On the view menu, choose sequence distances. How similar are these two proteins?

11. On the file menu, choose save, then give the file a name such as "protein alignment" and save it in your factor9 folder.

F. Construction of a Recombinant DNA Sequence.

1. Open the DNASTAR program EditSeq.

2. On the file menu, choose open, then select the PCR product sequence from your factor9 folder.

3. On the file menu, choose open, then select the pBSSKplus sequence from the VectorData folder. On the file menu, choose save as, and save it to your factor9 folder.

4. Select the PCR product sequence window, position the cursor before the first 2 T’s and add a C (this would have come from the primer during PCR). On the file menu, choose save.

5. Click and drag to select your entire PCR product sequence, on the edit menu, choose to lowercase, then again on the edit menu, choose copy.

6. Select the pBSSKplus sequence window, on the search menu, choose find, the enter the sequence "AAGCTT" (the HindIII recognition sequence) and click find next.

7. Position your cursor between the G and the C of the HindIII site on the vector, then on the edit menu, choose paste. On the file menu, choose save as, and save the sequence to your factor9 folder as "cloneA".

8. There is a 50% chance that the PCR product will be inserted in the orientation described above. There is also a 50% chance that it will be inserted in the reverse orientation. Select the PCR product window (your sequence should still be highlighted), then, on the goodies menu, choose reverse complement.

9. On the file menu of the new window, choose save and give it the filename "PCR product reverse".

10. Construct a recombinant plasmid with the PCR product reverse sequence as described above for the original PCR product and save it with the filename cloneB.

G. Mapping the Recombinant Plasmids

1. Open the DNASTAR program MapDraw, Click File, New, then double click on the "cloneA.seq" or "cloneB" file.

2. To obtain an uncluttered view of the sequence, it may be necessary to remove some of the features shown by altering the settings under the options menu. The goal is to have the sequence displayed as shown to the right.

• go to Enzyme Display, check none
• go to Reading Frames, check none go to Line Layout, make sure only DNA Ruler at the top and Space at the bottom are checked.

3. On the Enzyme menu, select new filter, then manual pick. Click and drag the following enzymes from the right to the left column of the "new pick filter" dialog box – BamHI, BstEII, ClaI, EcoRI, EcoRV, HindIII, KpnI, NdeI, PstI, PvuII, SalI, SmaI, XbaI, XhoI. Enter your name in the name box, click apply, then OK.

4. On the options menu, select enzyme display, then horizontal. Browse through the site and sequence display to identify the restriction enzyme recognition sites.

5. Click and drag to select the sequence between the two HindIII sites. On the features menu, choose new feature, enter the name "factor IX insert" in the subsequent dialog box, put a check mark in boxes below show on. Click on OK, then close the feature list box by clicking the X box in the upper right hand corner.

6. On the Map menu, select worksheet, examine the resulting graphic.

7. On the Map menu, select linear minimap, examine the resulting graphic. Use this map to determine the sizes of the fragments that would result from cutting you clone with various enzymes. Compare the results between cloneA and cloneB. How could you distinguish between the two possible orientations?

8. On the Map menu, select linear illustration, examine the resulting graphic.

9. On the Map menu, select circular illustration, then on the options menu, select drawing size. Change the width setting to 8 inches, click OK. On the options menu, select show actual size. Examine the resulting graphic.

10. On the file menu, choose save to save your map in your factor9 folder.

11. To save this graphic for inclusion in your lab report, we will do a screen grab. Display the graphic on your screen and press the "print screen" button on your keyboard.

12. Open the Microsoft PhotoEditor program. On the Edit menu, select paste as new image.

13. Click the dashed box button on the toolbar, draw a box around the graphic. On the image menu, select crop, then click OK.

14. On the file menu, select save as , then save the image as a jpg file, which can later be inserted into your lab report.

This page was created or last modified on 10/24/99 by Jeff Newman
and has been accessed  times since 10/24/99.

© 1998, 1999  Jeffrey D. Newman