No this blog is not a belated April fools joke… there really is a method to save thousands of dollars on microsatellite marker multiplex and genotyping! If you are just reading my blog for the first time, this is Part II following up on my last blog: How-to use microsatellites for population genetics, Part I: Study Design, DNA extraction, Microsatellite Marker Design/Outsourcing.
When I first set out to work with microsatellites, I was on a budget and I had never had experience with multiplex PCR, genotyping or fluorescent markers before- so there was definitely an uphill learning curve.
To start (assuming you already have your microsatellite markers- see previous blog for this)- the next step is to order your fluorescent markers to see how things work in multiplex PCR. FYI-Don’t order your Liz Size Standard until you are done troubleshooting and checking things on gels because it expires kinda quick! Plus it ships really fast (at least if you are in the USA).
What are fluorescent markers you ask? If you look at the microsatellite genotyping peaks above, the different color peaks correspond to the different fluorescent tails that are ‘attached’ to the microsatellite primers. FAM= blue, PET = red, VIC =green, and NED = yellow. This way, you can see the different colors and know what markers they correspond to. The orange peaks are from the 600 Liz size standard which enables you to actually calibrate the size of the peaks. This is very easy if you use a program like Geneious as they have a microsatellite plugin. I will detail more of the data handling in Part III of this blog series.
Why multiplex PCR? Simple.. it is faster (& cheaper if you troubleshoot things quick).
Many folks use multiplex genotyping, where you do singleplex PCR with all the separate microsatellite markers, and then you add for example 4 non-overlapping microsatellite marker amplified products (from your singleplex PCR) into a well together for downstream genotyping (more on that process later). This process saves money, since instead of genotyping a single marker for 1$.. you can genotype 4 markers for 1$! However- singleplex PCR takes forever if you have a lot of samples and markers!!!! If you have 400 individual DNA extracts, and each sample requires 10 markers of genotyping.. this means 4000 PCR REACTIONS- YIKES- that would result in carpal tunnel in a heartbeat!
Multiplex PCR in contrast allows you to add 2-4, (or more) microsatellite markers with fluorescent tails into the PCR mix, making sure that markers with the same fluorescent tails don’t overlap in size (ie- a FAM marker amplified product of 100-250 bp compared to another FAM marker amplified product of ~300-400 bp size range- should be fine to put together in a mixture). Whereas you don’t need to worry as much if they are similar size but have different fluorescent markers (such as FAM (blue) versus NED (yellow)). There are pull-up issues, and inhibition issues.. but that is why you will need to test everything out first anyhow before running your final assays.
As for myself, two papers were key in learning how to streamline microsatellite multiplex and genotyping: Blacket et al. 2012 and Culley et al. 2013. Both studies utilized four universal fluorescent tails (different ones in the different studies)- so that all you need to do is to add the non-fluorescent tail (just the ATCGs) of the corresponding fluorescent tail (the ATCGs + the FAM, VIC, NED or PET fluorescent marker) to your forward primer, and then use pig-tails on your reverse primers (such as GT, GTT, GTTT- depending on your reverse primer). Cullen et al. 2013 has an appendix which actually walks you through every step, including the reaction concentration of each Forward primer +tail, Reverse primer +pig-tail, and Fluorescent marker +tail in the final multiplex mix. I ended up using the four universal tails in Blacket et al. 2012, and then used Culley’s reaction mixes.
Below is a great pictorial image of how this works (from Blacket et al. 2012)
In addition to reading these papers (and their supplementary material) thoroughly, I recommend the following:
- Talk to as many people as you can before you start/ while you are getting started -you always learn fabulous tips and tricks as well as what not to do!
- Use the multiplex manager program -this will help you simulate what markers are compatible with each other based on the estimated product sizes, the melting temperatures (Tm) and the specific tails and flourophores you want to use. This will help you think about the different multiplex reactions that you can use.
- Order the Qiagen Multiplex Plus Kit– this will streamline everything! I accidentally ordered the regular multiplex kit.. which is an older version and slower- so I had to stick with it once I got started. However- the Plus version enables you to use a faster PCR protocol! This kit is the same thing as their old ‘Type-it kit”, just better.
- Order a set of just the universal tails without the fluorophores attached first, in addition to the forward primers with the universal tails, and the reverse primers with the pig tails. This will let you try running all the multiplex reactions out and test them on a gel to make sure you have everything working before you waste your precious flourophores which are expensive. This is also a cheap thing- 4x $6 max.. 24$ to try out a bunch of stuff before spending the big money is well worth it! By the way you will order all of your flourophores (reporter dyes) attached to the universal tail or the forward primer (the latter is a more expensive technique) from Thermo Fisher Scientific as they have patents on all of them except for FAM, which you can buy cheaper from Sigma or other companies (IDTdna, Elim BioPharm…etc)
- When you have everything working (bands are where you expect them to be, and no large gaps between bands which indicate that the msat markers are targeting multiple regions of the DNA sequence rather than one region)- Then order black, sterile micro centrifuge tubes (this link is just an example- but any sterile brand will work)- this will be what you make all your fluorescent primer ‘party’ mixes in, which will protect the fluorescence from degradation- preventing you from having to order more and save you money and time!
- Color code everything -from the tops of your individual fluorescent forward-reverse-fluorescent primer mixes (FRT: Forward-tail +Reverse-pigtail +Fluorophore-Tail) to your tubes with primer party mixes (all four FRT), to your multiplex pcr reaction mixes and to your excel files for the pcr-plates and genotyping plates.
- Be organized when you pipette things onto your pcr-plate, and into your genotyping plate, see below image on how I organize pipetting into a pcr-plate, with the samples in the large tubes being moved in the order that I add things to the plate. I close the lid of each tube and move it to the upper tray after I add it to the plate so I don’t lose my place. I also use my tips from the box in order so that I can look at my tip-box as well to see where I should be.
- Talk to the genotyping facility about if they permit a ‘troubleshooting’ run (free-of-charge) so that you can test the amount of final pcr product to add into each genotyping well. I used 0.5 ul pcr product (that had amplified products of four markers) with 11 ul genotyping mix (Liz size standard in Hi-di formamide)
Random Tips/Interesting findings:
- The Liz size-standard has strict “keep in dark and don’t-freeze’ instructions when it is shipped to you. Be sure to put it in the fridge at 4 degrees and NOT in the freezer! Unfortunately it comes in a styrofoam box w/ ice-packs with no-outside labels instructing to not freeze…and so the mailing department or your lab technician might accidentally put it in the freezer (speaking from personal experience…)- The company (Thermo Fisher Scientific) was very nice in shipping me a replacement because of this issue. However… due to this occurrence I had the opportunity to test whether or not the Liz -size standard would still work when frozen for 6 hours, and when frozen for 24 hours….Results: The size standard still works great when frozen for 6 hours and for 24 hours ! With that said.. obviously don’t purposely test this, but if it is accidentally frozen- chances are you are still ok!
- I also found that the Liz size-standard works great and is consistent for at least 2 months beyond its written expiry date…
- For both of these tests I had unexpired and unfrozen Liz-size standard to compare these tests to. No p-value available.. just my experience 😉
- As for the genotyping- I used less Liz Size Standard than recommended (and so did everyone that I talked to). My specific reaction mixes were the following: 0.5 ul PCR product + 0.5 ul Liz Size standard, and 10.5 ul Hi-Di Formamide per genotyping reaction well. I know a lot of folks that use 0.5 ul PCR product + 0.2 ul Liz-size standard+ 9.3 ul Hi-Di Formamide.. and they have great success as well. I tried the latter mix and it worked, but because my pcr products had such high fluorescence (and I was over troubleshooting my primer fluorophore mix concentrations- I decided to instead increase my size-standard so that I could better separate the noise from the signals). My genotyping mix and final primer ‘party’ mixes result in the initial genotype peaks image of this blog- so you can see what I mean by high sample peaks compared to the size-standard.
- As for the Hi-Di Formamide- I noticed that this does not have good results when you leave it in the fridge overnight and try to use it the next day for a second genotyping plate. However- I had good results with using Hi-Di Formamide that underwent 1-3 freeze-thaw cycles. Thus my advice is that you never leave it at room temperature or in the fridge if you have extra, but also to avoid too many freeze-thaws.
- Additionally- Im sure you will find this out- but NEVER freeze your pcr-products after the multiplex pcr with the flourescent markers, or after you add the liz-size standard and hi-di formamide. If you can’t get your samples to the genotyping facility right away, then be sure to do a quick denature (95 degrees for 5 min) post combining the pcr product w/ the Liz size standard and hi-di formamide and then just keep in the fridge (and in the dark!) until the next day or two.
Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.
Culley, T.M., Stamper, T.I., Stokes, R.L., Brzyski, J.R., Hardiman, N.A., Klooster, M.R., Merritt, B.J. 2013. An efficient technique for primer development and application that integrates fluorescent labeling and multiplex PCR. Appl Plant Sci 1.