Learning Exercise
Primer Design 2
In addition to alignment software, students use programs which will calculate Tm and formation of primer-dimers.
- Material:
- Bioinformatics and Molecular Biology
- Date last modified:
- November 9, 2000
- Created by:
- Scott Cooper
- License:
-
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Exercise
Purpose: to use a database to design specific PCR primers to amplify a region of DNA.
Go to Biology Workbench and log in.
Select Session Tools and then New Session.
Name the New Session Deer Bone
Next select Nucleic Tools and then Ndjinn (this is a search engine for DNA and protein sequences)
In the search window enter the following three ascession numbers separated by "or"
AF016978 OVU12869 BBU12864
Next scroll down and click the box next to the term GBMAM (GenBank Mammal)
When the three sequences appear, select Import Sequences
These three sequences will now be imported into your Deer Bone folder.
What region of DNA do these sequences correspond to? From which species?
Click on the boxes next to each sequence file and then select CLUSTALW
This program will align the three sequences.
Your assignment is to use this aligned sequence to design PCR primers which will amplify all three DNAs.
The Tm of the primers should be >50C. Also design the primers such that the PCR products from the two different species will be different sizes. This will allow for rapid identification of the species.
To check the Tm of primers use the program on the page PRIMER DESIGN in BioWeb.
To be turned in for credit:
q
Print out the report for the two primers you end up choosing, showing the Tm, length, etc.qTurn in a printout of the aligned sequences with the location of the two primers indicated.
qWhat sized PCR product will you generate in each species?