Learning Exercise
DNA Primer Design
A exercise on designing effective DNA primers.
This exercise asks students to design primers that would bind to two sequences for PCR amplification.
The students then use commercial programs to calculate the melting temperature (Tm) of each primer and to look for primer dimers.
This exercise asks students to design primers that would bind to two sequences for PCR amplification.
The students then use commercial programs to calculate the melting temperature (Tm) of each primer and to look for primer dimers.
Course: Molecular Biology
- Material:
- Bioinformatics and Molecular Biology
- Date last modified:
- September 28, 2000
- Created by:
- Scott Cooper
- License:
-
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Exercise
Design primers 15-25 bp long that will amplify both of the sequences below.
You may wish to use an Alignment program to identify conserved regions in both
sequences. These sites would be good targets for PCR primers to bind to both sequences.
Recall that the two primers need to bind to the target DNA such that the free 3'
ends of each primer point towards each other.
You may wish to review the rules used design primers.
After you have identified the sequence of your primers, check the primers with
the programs used to calculate melting temperature (Tm) and the formation of
primer dimers.
If the Tm is less than 55oC or bad hairpins or dimers form, try another region
of sequence.
>Paddlefish
CCTTGGCCTCTGCCTAATCACACAGATTCTAACAGGATTATTTCTCGCAATACACTACA
CAGCTGACATCTCAACAGCCTTCTCCTCCGTCGCCCACATCTGTCGAGATGTTAACTAC
GGATGACTAATTCGAAACATTCATGCAAACGGAGCCTCCTTTTTCTTCATCTGCCTCTAC
CTTCACGTAGCCCGAGGCATATACTATGGCTCATACCTCTACAAAGAAACCTGAAACAT
CGGAGTAGTTCTCCTACTCCTAACTATAATAACCGCCTTCGTAGGATATGTGCTCCCATG
AGGACAGATATCCTTCTGAGGAGCCACCGTAATTACCAACCTTCTTTCCGCCTTCCCCTA
CATCGGGGACACCCTAGTACAATGAATCTGAGGTGGTTTCTCAGTAGACAACGCCACCC
TAACC
>Shovenose Sturgeon
CCTAGGCCTCTGCCTTATTACACAAATCTTAACAGGACTATTTCTTGCAATACACTACACA
GCTGACATTTCAACAGCCTTCTCCTCCGTCGCCCACATCTGCCGAGACGTAAACTACGGG
TGACTAATCCGAAACGTCCACGCAAATGGCGCCTCCTTCTTCTTTATCTGCTTGTACCTTCA
CGTCGCACGAGGTATATACTACGGCTCCTACCTCCAAAAAGAAACCTGAAACATCGGAGT
AGTCCTCTTACTCCTCACCATAATAACCGCCTTCGTAGGCTATGTACTGCCCTGAGGACAA
ATATCATTTTGAGGGGCAACCGTAATCACTAACCTCCTTTCCGCCTTCCCGTACATCGGCG
ACACATTAGTGCAATGAATCTGAGGCGGCTTTTCAGTC
You may wish to use an Alignment program to identify conserved regions in both
sequences. These sites would be good targets for PCR primers to bind to both sequences.
Recall that the two primers need to bind to the target DNA such that the free 3'
ends of each primer point towards each other.
You may wish to review the rules used design primers.
After you have identified the sequence of your primers, check the primers with
the programs used to calculate melting temperature (Tm) and the formation of
primer dimers.
If the Tm is less than 55oC or bad hairpins or dimers form, try another region
of sequence.
>Paddlefish
CCTTGGCCTCTGCCTAATCACACAGATTCTAACAGGATTATTTCTCGCAATACACTACA
CAGCTGACATCTCAACAGCCTTCTCCTCCGTCGCCCACATCTGTCGAGATGTTAACTAC
GGATGACTAATTCGAAACATTCATGCAAACGGAGCCTCCTTTTTCTTCATCTGCCTCTAC
CTTCACGTAGCCCGAGGCATATACTATGGCTCATACCTCTACAAAGAAACCTGAAACAT
CGGAGTAGTTCTCCTACTCCTAACTATAATAACCGCCTTCGTAGGATATGTGCTCCCATG
AGGACAGATATCCTTCTGAGGAGCCACCGTAATTACCAACCTTCTTTCCGCCTTCCCCTA
CATCGGGGACACCCTAGTACAATGAATCTGAGGTGGTTTCTCAGTAGACAACGCCACCC
TAACC
>Shovenose Sturgeon
CCTAGGCCTCTGCCTTATTACACAAATCTTAACAGGACTATTTCTTGCAATACACTACACA
GCTGACATTTCAACAGCCTTCTCCTCCGTCGCCCACATCTGCCGAGACGTAAACTACGGG
TGACTAATCCGAAACGTCCACGCAAATGGCGCCTCCTTCTTCTTTATCTGCTTGTACCTTCA
CGTCGCACGAGGTATATACTACGGCTCCTACCTCCAAAAAGAAACCTGAAACATCGGAGT
AGTCCTCTTACTCCTCACCATAATAACCGCCTTCGTAGGCTATGTACTGCCCTGAGGACAA
ATATCATTTTGAGGGGCAACCGTAATCACTAACCTCCTTTCCGCCTTCCCGTACATCGGCG
ACACATTAGTGCAATGAATCTGAGGCGGCTTTTCAGTC