week 6 homework

Please send me your answers by email. You can either create a new file, or download the ms word file and type in your answers.

For this exercise I strongly recommend using MacVector (at least, for cloning in silico). You can do it online, but cutting and pasting the nucleic acid files at the correct locations will be very difficult.

I have found some free DNA sequence manipulation programs for the PC at the link listed below, but I have no idea how good or user-friendly they are.

4) Now analyze this sequence using Sixframe at Biology workbench

5) How easy was it to interpret the output?

6) Which frame gave the longest ORF, and how long was it?

7)Now try the Danish site, http://www.cbs.dtu.dk/services/NetStart/

8) Now analyze the sequence with MacVector or your PC program

MacVector instructions
- Start MacVector, and under the file menu choose NEW | Nucleic Acid (You may need to click on the MacVector slide that comes up when you first start it before you can do anything else).
- Copy the Cuphea lanceolata DNA sequence and paste it into the new file
- Save the file into the USERS Folder on the hard disk, then select ANALYZE|OPEN READING FRAME
- Select start/stop codons and set min# amino acids = 100
- Select "list ORFs by position" and attach the output to your homework.
- How many ORFs did you find?
- Where does the longest ORF start and stop?
- Is this the same place as the longest ORF found by GORF at NCBI?
If you use a PC program, perform a similar analysis

Part III: Translation

1.Translate the longest ORF using any program or site you choose and the output to your homework

MacVector instructions
- Open the Cuphea lanceolata file
- select ANALYZE| translation
- Enter the start and stop coordinates for your longest ORF in the "Segment to translate" window. Be sure to end with a semicolon! E.g 35/998;
- Click the "create new protein" button, give your baby a name, then click OK
- attach a screenshot of the output to your homework

Part IV: Proteolytic digestion

6. Now try designing two primers that you can use to amplify the coding sequence either using Primer 3 or Mac Vector.

MacVector users, note that you can also design degenerate primers for a protein, or a consensus from a ClustalW alignment, by selecting that window (e.g. the consensus ) and choosing ANALYZE| reverse translation, then select the "probe list" button.

Your mission is to subclone the coding sequence of Cuphea lanceolata DNA sequence into the pBS plasmid in a way that will function in bacteria. This means that you must design PCR primers that will amplify the coding sequence including the start and stop codons. You will also need to add suitable restriction sites to clone it into pBS.

1. Identify two restriction enzymes that cut the bluescript polylinker (see the figure in this week's lecture) but do not cut the Cuphea lanceolata DNA sequence

2. Design a PCR primer that will anneal to the 5' end of the Cuphea lanceolata DNA either 5' to or including the ATG codon. The melting temperature for the primer must be ~ 60 C.

3. Add a suitable restriction site at the 5' end of this primer (i.e., one of the enzymes identified in step VII.1)

4. Design a PCR primer that will anneal to the 3' end of the Cuphea lanceolata DNA either 3' to or including the stop codon. The melting temperature for the primer must be ~ 60 C.

5. Add a suitable restriction site at the 5' end of this primer (i.e., the site recognized by the other enzyme identified in step VII.1).Note, the 5' end of this primer is the reverse and complement of the 3' end of the gene!

8. Attach the sequence of your clone. Be sure to print the pBS sequence in upper case and your cloned sequence in lower case, so that it is obvious where they change.

9. Attach a map of the predicted plasmid showing enzymes that cut it once and twice.

bioinformatics

Home syllabus general information homework lectures websites Will Terzaghi's Homepage Membership Login				week 6 homework Restriction analysis and cloning in silico Due February 22, 2003 Please send me your answers by email. You can either create a new file, or download the ms word file and type in your answers. "week6homework.doc" For this exercise I strongly recommend using MacVector (at least, for cloning in silico). You can do it online, but cutting and pasting the nucleic acid files at the correct locations will be very difficult. I have found some free DNA sequence manipulation programs for the PC at the link listed below, but I have no idea how good or user-friendly they are. http://www.cellbiol.com/modules.php?op=modload&name=Downloads&file=index&req=viewsdownload&sid=5 Part I: Restriction analysis Go to "week 5 websites" and copy the Cuphea lanceolata DNA sequence. Go to Webcutter (http://www.firstmarket.com/cutter/cut2.html) and load this DNA sequence. How many enzymes did not cut this sequence (be sure that for all these analyses you specify "all enzymes")? How many enzymes cut this sequence once? How many enzymes cut this sequence twice? How many enzymes cut this sequence between 3 and 5 times? Go to the biologist's workbench (http://workbench.sdsc.edu/) and analyze this sequence with TACG (It should already be loaded there from last week). Be sure that for all these analyses you specify "all enzymes." How many enzymes did not cut this sequence? How many enzymes cut this sequence once? How many enzymes cut this sequence twice? How many enzymes cut this sequence between 3 and 5 times? Now analyze this sequence with either MacVector or one of the PC programs. How many enzymes did notcut this sequence? How many enzymes cut this sequence once? How many enzymes cut this sequence twice? How many enzymes cut this sequence between 3 and 5 times? Which of these three programs/websites was easiest to use? Which of these three programs/websites gave you the most useful information? Which of these three programs/websites would you use for your research? Part II: Finding ORFs 1) GO to GORF (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) paste in the Cuphea lanceolata DNA sequence, then analyze it. Attach the output to your homework 2) Now try the EMBL site (http://www2.ebi.ac.uk/translate/) 3)Did your procedure give you the same protein sequence? What is wrong? How can you fix it? 4) Now analyze this sequence using Sixframe at Biology workbench 5) How easy was it to interpret the output? 6) Which frame gave the longest ORF, and how long was it? 7)Now try the Danish site, http://www.cbs.dtu.dk/services/NetStart/ Click on Abstract. Why is prediction of start sites non-trivial? How well does NetStart predict start codons in Arabidopsis? Now paste your Cuphea lanceolata DNA sequence into the submission window, select A..thaliana then click "submit" how many start sites did it find? Which was the most likely? 8) Now analyze the sequence with MacVector or your PC program MacVector instructions Start MacVector, and under the file menu choose NEW \| Nucleic Acid (You may need to click on the MacVector slide that comes up when you first start it before you can do anything else). Copy the Cuphea lanceolata DNA sequence and paste it into the new file Save the file into the USERS Folder on the hard disk, then select ANALYZE\|OPEN READING FRAME Select start/stop codons and set min# amino acids = 100 Select "list ORFs by position" and attach the output to your homework. How many ORFs did you find? Where does the longest ORF start and stop? Is this the same place as the longest ORF found by GORF at NCBI? If you use a PC program, perform a similar analysis Part III: Translation 1.Translate the longest ORF using any program or site you choose and the output to your homework MacVector instructions Open the Cuphea lanceolata file select ANALYZE\| translation Enter the start and stop coordinates for your longest ORF in the "Segment to translate" window. Be sure to end with a semicolon! E.g 35/998; Click the "create new protein" button, give your baby a name, then click OK attach a screenshot of the output to your homework Part IV: Proteolytic digestion 1.Copy the translated sequence and paste it into either MacVector or http://us.expasy.org/cgi-bin/peptidecutter/peptidecutter.pl 2.Analyze it with all of the enzymes and chemicals available 3. How many enzymes and chemicals cut it? 4.Which cut it the most? 5. How many did not cut it? MacVector instructions Click on the file of your new protein that you created in the previous step (it should already be in front) select ANALYZE\| Proteolytic Enzyme, then search using all enzymes and click OK in the next window, select "List cutters by name" and "list non-cutters," then click OK and attach the output to your homework Part V: Designing Primers Try designing two primers that you can use to study the cuphea lanceolata gene. First use the biologist's workbench (http://workbench.sdsc.edu/) PRIMER3, and the default settings. (alternatively, use http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi ) Where does the optimal left primer start? How long is it? What is its melting temperature? Where does the optimal right primer start? How long is it? What is its melting temperature? Where does the second best left primer start? How long is it? What is its melting temperature? Where does the second best right primer start? How long is it? What is its melting temperature? 6. Now try designing two primers that you can use to amplify the coding sequence either using Primer 3 or Mac Vector. Primer3 instructions Set the target region as starting at the beginning and stopping at the end of your longest ORF Where does the new optimal left primer start? How long is it? What is its melting temperature? Where does the new optimal right primer start? How long is it? What is its melting temperature? MacVector.instructions Open the Cuphea lanceolata DNA file Under the "Analyze" menu select Primers \| PCR primer pairs Under "Scan Criteria" select "find pairs by specifying" two flanking regions., then enter 1 to the start of your ORF for the forward primer and the end of your ORF to the end of your sequence for the backwards primer Leave the other settings alone and click OK Where does the optimal forward primer start? How long is it? What is its melting temperature? Where does the optimal backwards primer start? How long is it? What is its melting temperature? Part VI: Designing Degenerate Primers 1.Copy the "Clustal query" sequences from week 4 websites, then go to the BLOCKS website and paste them into BLOCKMAKER. http://blocks.fhcrc.org/blocks/blockmkr/make_blocks.html 2. When you get your BLOCKS output (3 lines below the Proweb tree viewer button) you will see Primers: CODEHOP 3. Click on "About Codehop" What is the program intended for? What part of each primer is degenerate? Scroll down to "Terms and parameters." What does degeneracy measure? Scroll down to "Basic tips." What do they recommend you do if you do not get predictions? 4. Return to your "Blockmaker Results" window and select Codehop. Leave the settings alone and click "look for primers" Copy the primers that you find and attach them to your homework Return to Codehop, and this time set the degeneracy to 512 and the temperature to 40 Now how many primers to you get? MacVector users, note that you can also design degenerate primers for a protein, or a consensus from a ClustalW alignment, by selecting that window (e.g. the consensus ) and choosing ANALYZE\| reverse translation, then select the "probe list" button. Part VII: Cloning in silico Your mission is to subclone the coding sequence of Cuphea lanceolata DNA sequence into the pBS plasmid in a way that will function in bacteria. This means that you must design PCR primers that will amplify the coding sequence including the start and stop codons. You will also need to add suitable restriction sites to clone it into pBS. 1. Identify two restriction enzymes that cut the bluescript polylinker (see the figure in this week's lecture) but do not cut the Cuphea lanceolata DNA sequence 2. Design a PCR primer that will anneal to the 5' end of the Cuphea lanceolata DNA either 5' to or including the ATG codon. The melting temperature for the primer must be ~ 60 C. 3. Add a suitable restriction site at the 5' end of this primer (i.e., one of the enzymes identified in step VII.1) 4. Design a PCR primer that will anneal to the 3' end of the Cuphea lanceolata DNA either 3' to or including the stop codon. The melting temperature for the primer must be ~ 60 C. 5. Add a suitable restriction site at the 5' end of this primer (i.e., the site recognized by the other enzyme identified in step VII.1).Note, the 5' end of this primer is the reverse and complement of the 3' end of the gene! 6. Simulate your PCR amplification. Copy the target sequence from the first base of the forward primer to the last base the backwards primer anneals to and paste it into a new file. Add the 5' restriction site to the 5' end of this sequence, and the 3' restriction site to the 3' end. This is the actual piece of DNA that would be generated if you were to perform PCR on Cuphea lanceolata DNA using these two primers! 7. Now insert this sequence into pBS. Copy the entire sequence highlight pBS between the 5' restriction site and the 3' restriction site Paste the sequence into pBS. You should replace the .pBS sequence between the two sites with Cuphea lanceolata sequence The plasmid should become about a thousand basepairs larger The restriction sites between your 5'and 3' site should disappear. 8. Attach the sequence of your clone. Be sure to print the pBS sequence in upper case and your cloned sequence in lower case, so that it is obvious where they change. 9. Attach a map of the predicted plasmid showing enzymes that cut it once and twice.
Last update: Saturday, February 22, 2003 at 2:02:11 PM.