Creating Primers for PCR
Overview: You will use a reference sequence of the D-loop of human mtDNA to determine
what primers to use for PCR amplification of this region of DNA. Primers are used to target
the desired sequence so the polymerase knows were to attach to begin replication.
1. Go to Primer 3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi).
2. Once you get to the site, the first thing you will need to do is insert the DNA
sequence you want to find primers for. We are interested in the HVR1 region of the
D-loop of human mtDNA. To find this region, go to Bioservers
(http://www.bioservers.org/bioserver/).
3. Under the “Sequence Server” column, click “Enter”.
4. Along the top center of the page, click on “Manage Groups”, and under the “Sequence
sources” pull-down menu choose “Mitochondrial Genomes”. Choose “Human
Mitochondrial D-loop sequences” and click “ok”.
5. In the pull-down menu, make sure the HVR1 section is the one selected, then click
“Open”. In the box is the sequence for HVR1. We want to make sure the DNA
fragment we amplify contains as much of this sequence as possible, so we’re going
to look up the sequence of mtDNA a little further out from HVR1 as well.
a. Note that the HVR1 sequence is within the basepairs 16020 and 16400 of
human mtDNA.
b. Go to http://www.ncbi.nlm.nih.gov/nuccore/251831106 to find the
Cambridge Reference Sequence for the entire human mtDNA sequence.
c. Find an area of the sequence ~100 basepairs (you can play around with the
starting area to see how the primers may differ if you choose) from the start
of the HVR1 sequence and copy the sequence starting from that location to
the end of the mtDNA sequence.
d. Copy and paste this into a Word document to clean it up (remove the
basepair numbers so all you have left is the nucleotide sequence).
6. Go back to your Primer 3 page and insert this sequence into the large box at the top
of the page. Set the product minimum size to 400, the product optimum size to 500,
and the product maximum size to 1000. Click “Pick Primers”.
*Note: The general size of sequences desiring to be amplified by standard
PCR is around 500 bp. Things to keep in mind when designing a primer:
- Primer Melting Temperature (Tm) – this is the temperature at
which half of the DNA will dissociate to become single stranded.
Tm should be 52-58C.
- GC Content (give as a % of the total bases) – number of guanine
and cytosine bases in the primer; should be 40-60%.
- GC Clamp – having guanine and cytosine nucleotides within the
last 5 bases of the 3’ end of the primer helps with specific pairing
due to the high affinity of guanine and cytosine for each other; no
more than 3 G’s or C’s should be within the last 5 bases of the 3’
end
- Repeats (ie. ATATAT) and Runs (GGGGG) – should be avoided
because they can lead to mismatching; should have no more than 4
di-nucleotide repeats and no more than 4 basepair runs
-
Primer Pair Tm Mismatch Calculation – the difference between the
Tm for each primer should be no greater than 5C as such a
difference could lead to no amplification.
7. Look at the different primer options provided and fill out the table below. (Play
around with the product size length to see if you get any other options as well.)
Left (Forward) Primer
Right (Reverse) Primer
Sequence
Size (bp)
Tm
Left
Tm
Right
GC
Left
GC
Right
8. Pick your top pair of primers. Write them below and explain why you chose that
pair.
9. Once given the PCR Amplification Procedure, write the sequences of the actual
primers that were used below and explain why they are good primers.