This exercise shows you how to use bioinformatics tools available on the internet to design sequencing primers. Primers are oligonucleotides used to direct DNA polymerase to a specific site on a DNA template. The enzymatic activity of the DNA polymerase extends the primer in the 3' direction; the sequence of the synthesized DNA is governed by the template. To obtain the sequence of a DNA sample, you would typically send the sample to a facility that specializes in DNA sequencing. However, you have to provide the facility with specific sequencing primers that are appropriate for your DNA sample. Here, we will discuss the process of designing sequencing primers step-by-step, using a specific example. The goal of the exercise is that you learn how to design sequencing primers on your own (there is a homework problem...) and understand the underlying concepts. There is a video demonstration showing you how to do this exercise, and some slides reviewing the basics of DNA:DNA interactions and DNA polymerase biology.
Today's date
Our task
A coworker asks us to check the sequence of an expression plasmid. The plasmid backbone is pET23d, into which the Mfd gene has been inserted, yielding the recombinant plasmid pET23d-Mfd. To study the effect of amino acid Arg902 on the ATPase activity of Mfd, our coworker has used site-directed mutagenesis to change the codon of residue 902 to code for alanine instead of arginine. Our job is to verify the sequence around the mutation, and also to sequence the entire gene plus promoter region to make sure there aren't any secondary mutations introduced during the site-directed mutagenesis. The sequence of the expression plasmid pET23dMfd (PlasmidA) and the desired mutated sequence, pet23dMfd-R902A (PlasmidB), are available.
Worksheet
Where on the plasmid is the mutation (compare the two plasmid sequences) ?
Where on the plasmid do we want the primer to bind?
On which strand do we want the primer to bind?
top
bottom
What is the melting temperature of the primer-template duplex?
deg C
What is the melting temperature of the most stable secondary structure of the primer?
deg C
What is the melting temperature of the second-best binding site on the plasmid?
deg C
What is the sequence of your primer?
5'-
-3'
Review questions
How can you tell whether a primer is sufficiently long to work in a sequencing reaction?
How can you test whether a primer will bind specifically to the intended site on the template?
Why should you avoid primers that contain self-complementary sequences?
How can you use the concept of thermodynamic vs. kinetic control to choose experimental conditions that favor primer-template hybridization over template reannealing?
Sketch of the experimental protocol
Before you design the primers, you should know the principles of classical dideoxy sequencing. Nowadays, a single extension reaction is usually preformed, using four differently fluorescing ddNTPs and the protocol sketched below.
Mix template, primer, DNA polymerase, dNTPs and fluorescent ddNTP analogs
Incubate at 95 deg Celsius for 30 seconds (Purpose? )
Rapidly cool to 50 deg Celsius and incubate for 10 seconds (Purpose? )
Keep at 60 deg Celsius for 4 minutes (Purpose? )
Repeat steps 2.-4. to run multiple extension reactions
Separate the reaction mixture by capillary electrophoresis and detect fluorescence at four different wavelengths
Blast2Sequence (similarities and differences between 2 sequences)
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Your task, after class
Design a sequencing primer to verify the mutation on the other strand (i.e. if the bottom strand was sequenced, now sequence the top strand and vice versa)
