Download Mulitplexing primer sets

Fragment Analysis - Multiplexing Primer Sets
Preparative work in multiplexing primer sets for genotyping experiments can be a great cost savings. The
preparative work begins with BOTH the forward and reverse primers untagged and allows for the
optimization of the primer sets and panel prior to purchasing the more expensive tagged forward primers.
Begin by selecting loci from previous work or literature. If you have worked with the primer sets already you
are a step ahead. If you have never worked with the primer sets than the preparative testing is invaluable.
General Steps:
1.) Design and order untagged F&R primers for each locus. In designing and selecting primers, work to
have the primers annealing Tm as close as possible within each primer set and also between the primer
sets. This will make the multiplexing much easier.
2.) Isolate your genomic DNA using whatever protocol works well for your starting material. The one
important aspect to the DNA preparation is to be sure the final clean DNA is in water. Most kits will have
a note at the elution step telling you that you can substitute water for the elution buffer that came with the
kit. If you are concerned this will affect your recovery contact the vendor for the kit and discuss yield with
water versus the elution buffer. But the most important point is that you NOT use a buffer if it contains
EDTA as this will affect Taq activity and reduce the amplicons you will be generating later.
3.) Normalize your template DNA. I emphasize this step because it is the one step everyone likes to
skip, but it is vital to ending up with good data. So start now and normalize your samples. For this you
need to quantify the DNA the genomic DNA you isolated and then prepare a plate where all of the DNA
samples are set to the same concentration. At NGC we set all template DNA to 5ng/ul and then use 4ul
(=20ng) per PCR. If you skip this step your data will be difficult to call as some samples will give very
low signal amplicons while others will give off-scale signal amplicons. Also, at this point in time you will
hopefully have 6-10 samples with good yields and you can use that extra DNA in the following two steps.
4.) Once your primers arrive, resuspend them as suggested below and prepare F&R primer mix for each
locus:
The paperwork will give you nmoles of DNA and you will resuspend accordingly. For example,
25.67nmoles of DNA you would resuspend in 257ul of TE = 100uM.
Tris/EDTA (TE) is 100mM Tris-Cl (pH74.) and 10mM EDTA (pH8.0)
Now, from this point on dilute using water so that you begin to dilute the EDTA used above.
Prepare 50uM F&R mix for each new primer set simply mix equal volumes of the forward and
reverse primers which are 100uM. So, combine 20ul of the forward (100um) with 20ul of the reverse
(100um) and you end up with F&R at 50uM for each primer.
Store ALL of the primers and primer mixes at -20C.
5.) If you have never run the primers before you need to begin by running each primer set individually to
check the amplicon’s sizes and fidelity of the primers. For this you want to prepare a small aliquot of the
F&R primer mix diluted to 2uM. Use the 2uM F&R mix and run a PCR using the optimal annealing Tm
for the primer set and some of your genomic template DNA (*see Taq suggestion in step 4). A good
generic thermocycler program to begin with is as follows:
Step
Suggested PCR Program
Temp in C
Time
1
95.0
15m
2
95.0
30s
3
Annealing Tm
45s
4
72.0
30s
5
back to step 2 34 cycles
6
72.0
45m
7
10.0
forever
Run the PCR products on an agarose gel and observe the size of the amplicons. Hopefully you have
only 1 or 2 amplicons. If there are more than 2 or if there is a great deal of smearing you will want to
work with that primer set, optimizing the thermocycler conditions and Tm until you can generate a clean
PCR with just 1 or 2 amplicons. If optimizing the thermocycler conditions does not help then you have
two options for that locus: redesign the primers or remove the primer set from those you will be working
with.
6.) Once you feel good about the loci you are working with you can design the panels. You need the
optimized PCR annealing temperatures and product sizes for this step. Combine the primer sets with
the same or similar annealing Tm in a panel. Hopefully the product sizes vary somewhat; but they can
be close in size. You may have more than 4 primer sets per panel (see step 6 below), but pay attention
to the product sizes. Once you have a panel designed on paper, prepare a panel mix beginning with
your 50uM F&R untagged primer mixes. Use 1ul of each 50uM F&R mix and then bring the final volume
for the panel to 25ul. For example, if you have 4 primers sets in the panel, you add 1ul of each 50uM
F&R mix for each primer set and then bring to a final 25ul volume with 21ul sterile water. The primers in
the panel mix are now at 2uM. Use the panel mix to run a PCR using the optimal annealing Tm for the
panel and with some of your genomic template DNA.
We at NGC have found that using Qiagen’s Multiplex PCR mix (Qiagen catalog #206145 or #206143) is
the best Taq for running multiplexed PCR. We recommend that for all the optimizing and then the run
that you have with the tagged panels. Here at NGC we run 10ul PCR using the Qiagen Multiplex master
mix as follows:
1sample
5 ul Qiagen MasterMix
1 ul primer mix
6 ul
add 4 ul 5ng/ul template DNA
Run the PCR on a gel and observe the size of the amplicons. Are all the expected amplicons present?
Some may not be as bright as others, but that can be remedied. If one of the primer sets has dropped
out it may be that primer set simply will not work with mix of primers in the panel. In that case you may
wish to move the primer set to a different panel. You can also play with the annealing Tm of the PCR for
the panel and see that helps. Finally, if one or more primer sets generates amplicons in the panel, but at
a decreased intensity, you can tweak the panel mix. Make up a fresh panel mix and use 2ul of the 50uM
F&R primer set rather than 1ul so that you have more of the primer set present in the panel.
7.) When you have panels running that give good results using gels to observe the results you are ready to
assign tags to the forward primers of each primer set. The Nevada Genomics Center 3730 run with
Filter set G5 so you can use dyes from ABI Dye Set DS33:
a. Red tag – PET
b. Blue tag – 6FAM
c. Yellow tag – NED
d. Green tag – VIC
Assign tags so that any primer sets that are the similar in size have different tags allowing them to be
distinguished. Keep in mind that hopefully you will have variation in the amplicon sizes for each locus.
You can have more than 4 primer sets per panel depending upon the sizes of the amplicons because
you can tag a locus with amplicons running ~200bp the same color as a second locus that has
amplicons running ~350bp.
8.) Order the tagged forward primers. When they arrive resuspend the stock aliquots of primer in TE and
store them in the freezer. Life Technology’s tagged primers always come with 10,000picomoles =
10nmoles. Resuspend the 10,000picomoles in 100ul TE = 100uM. You already have your reverse
untagged primers at 100uM.
Prepare 50uM F&R mix for each new primer set simply mix equal volumes of the forward and
reverse primers which are 100uM. So, combine 20ul of the forward (100um) with 20ul of the reverse
(100um) and you end up with F&R at 50uM for each primer.
Store ALL of the primers and primer mixes at -20C.
Prepare the multiplexed panel using the 50uM F&R with tagged forward primers. Run PCR using the
optimized conditions you generated in step 4 above. Send the PCR samples to the Nevada Genomics
Center and we will determine the optimal dilution to be used in running the samples in the 3730. The
samples will then be run with the LIZ ladder, a tagged DNA ladder used to call the sizes of the
amplicons. Your data for that run will be available on the NGC dnaTools server and you can download
the data and analyze it using GeneMapper or PeakScanner software.