PCR PROTOCOL

Primer testing protocols (See menu at left) for users (February 2008)

It has been brought to our attention that there might be some confusion with the primer testing protocol due to the fact that Takara sells a 10X Ex-Taq buffer that is Mg+2-free in addtion to the 10X buffer that contains 20 mM MgCl2.

In the protocols described in this section, the 10X Ex-taq buffer we use has 20 mM MgCl2. We add more MgCl2 to the reaction so that the final Mg+2 concentration is 2.7 mM. The Mg+2 concentration has been optimized for PCR with the IRD primers.

1. Introduction

     Primer testing by users is not a requirement of the Maize TILLING Project (MTP), MTP will "Pre-Screen" all primers to determine if they are suitable for TILLING. Our Pre-Screen is simply using your primers to amplify the target in gDNA from our TILLING inbreds (B73 and W22), analyzing the product on an agarose gel, and sequencing the PCR product. We want to confirm that the primers amplify a product of the expected size and that the single primers on their own do not produce any major products. The sequence of the B73 and W22 PCR products will serve as a reference when we sequence verify each putative mutation that we find in TILLING and when SIFT evaluates the mutation's effect on the protein. It is very important that the PCR product sequences cleanly. Runs of a single nucleotide, ~ 8 bases or more, often result in a sequence chromatogram that has 2 traces, one of which is offset by a base. These chromatograms are difficult to interpret in the context of verifying TILLING mutations.

Why would a user choose to test primers before placing an order if MTP is going to screen them anyway?

1) It will increase the likelihood that the primers pass the "Pre-Screen" the first time through.

     It takes MTP approximately 2 weeks to do the primer test and analyze the data. We will re-test a primer set if we are not satisfied with the first set of data, thereby adding at least another 2 weeks to the process. If you send us a primer set that you have tested under our conditions, most likely it will pass without out any problems. Please note, if your target is > 55% GC, the primer pre-screen may take longer because we automatically test these primers with TMSO (tetramethylene sulfoxide).

2) You are only allowed one "active" set of primers for each request.

     MTP will only screen one set of primers per target at a time. If your primers do not work well the first time, we will repeat the test one or two more times to be sure of the results. You must wait until we fail your original primer order before submitting the new primer order.

3) You can test several different primers and/or primers for several regions of their gene target at once to determine which set works best under our PCR conditions.

     This may be especially useful for GC-rich targets. We have found that GC-rich targets (PCR product > 55% GC) do not amplify well under our TILLING PCR conditions and require adding TMSO to the PCR which affects the TILLING gel data quality. We encourage our users with GC-rich targets to test several sets of primers with and without TMSO to determine which set works best. Similarly, if you have a duplicated gene, pre-testing the primers will allow you to verify that the correct target is being amplified.

     All orders placed with MTP must be initiated through CODDLe. You cannot place an order with simply the primer sequences alone, no exceptions. A variety of information is collected and stored in our database at the time an order is placed including the gene model and scoring information for the number of possible non-silent mutations in the target. Furthermore, use of our ordering system ensures primers are designed with parameters suitable for our TILLING assays. Our PCR conditions are unusual because they have been optimized for the IRD-labeled primers that we use in the TILLING PCRs.

     We have revised and clarified the PCR protocols and they can now be downloaded as a PDF files by clicking here (link). The file have protocols for PCR with and without TMSO and also for sequencing the PCR products.

     We have posted a "CODDLe Tutorial" that explains various features of CODDLe and the parameters that the user can change. CODDLe will suggest a minimum of 5 primer sets for your target, but you can increase this number. We use gDNA prepped in the same manner as the DNA used for TILLING, however you can use any PCR-quality B73 and W22 gDNA. Please try to select a primer that works on both B73 and W22 gDNA in order to maximize the number of mutant lines that we can screen. If you are having trouble designing primers or using CODDLe, go to the CODDLe tutorial and FAQ links. If your still need help, send an email to "maizetilling@purdue.edu" and in your correspondence please include the genomic DNA sequence and the cDNA and/or protein sequence and any CODDLe outputs you have generated.

     Some users have e-mailed MTP their data and asked for comments before submitting the order. We will gladly give you our opinion based on the data you send, but remember MTP will still repeat the Pre-Screen to ensure that the primers work in our hands and in our thermocyclers. We have found that > 90 % of the primers that have passed the Pre-Screen have produced good quality TILLING gels. Even though the Pre-Screen introduces a few extra steps to the process, the success rate at the TILLING stage makes it worth the time and effort.

2. Primer test protocol

Please read the "How to TILL" section before starting CODDLe . We also have a CODDLe tutorial that should answer most of the common questions. Our PCR and DNA sequencing protocols are available as a downloadable .pdf files

2.1 Primers to test

Once you have gone through CODDLe/Primer3, click "display this pair of primers" for the set of primers you want to test. You will get a page showing the gene model, protein homology models and information on the percent of different types of mutations predicted to be found. At the bottom of this page you will find an "order TILLING of this region" link. Print out this page and save this file as an html document. If you decide to use these primers for TILLING, just re-open the html file and click on the "order TILLING" button.

2.2 Testing primers for robust PCR amplification

Our TILLING populations are in the B73 and W22 backgrounds. If possible, test primers with gDNA from both inbreds. Note that MTP cannot supply genomic DNA. Our PCR protocols include a sample layout for testing several primers sets in a 96 well microtiter plate, PCR cocktails with and without TMSO and DNA sequencing. The cocktail mixes are for 100 reactions, but can be scaled down. Yes, our PCR conditions are unusual, but they do work for the IRD-labeled primers. The final concentration of Ex-Taq buffer is 0.5X and the MgCl2 concentration is 2.4 mM. The final volume of each reaction is 10 microliters. For each reaction, we use:

- 5.0 uL of genomic DNA (gDNA) at a concentration of 0.9 ng/mL (4.5 ng gDNA total),
- 0.3 uL of primers (working concentration 10 micromolar)
- 4.7 uL of PCR cocktail mix

We recommend doing the following reactions (see Protocol):

B73     L+R primers      (in duplicate)
W22    L+R primers      (in duplicate)
TE       L+R primers
Water  L+R primers      (optional)
B73     Left primer only
W22    Left primer only
B73     Right primer only
W22    Right primer only

Why do we include the single primer controls?

     When we do the TILLING reactions with the IRD-labeled dyes, the efficiency of the PCR is reduced. We have some evidence that a primer that generates PCR products on its own may not be a good TILLING primer. When such primers are used for TILLING, the TILLING gels tend to have non-specific bands, there is a reduction in the amount of full-length product and the gels are difficult to interpret. If the single primer control just has one or two low intensity bands, it is probably fine. We are more concerned about a primer that generates multiple products and where at least one of them is quite abundant.

Thermocycler Profile:

95 oC  2 minutes

Loop 1: 8 cycles
   94 oC 20 seconds
   73 oC 30 seconds
   -1.0 oC per cycle increment
   Ramp to 72 oC at 0.50 oC /second
   72 oC 1 minute

Loop 2: 45 cycles
   94 oC 20 seconds
   65 oC 30 seconds
   Ramp to 72 oC at 0.50 oC /second
   72 oC 1 minute

Final extension at 72 oC for 5 minutes
Hold (4 - 10 oC)

3. Analysis of PCR products

3.1 Agarose Gel analysis

     Take 2 µl of sample from the PCR reactions and load on a 1% agarose gel. Include on this gel 4 µl of Low DNA Mass Ladder (Invitrogen) or an analogous quantitative ladder. We also recommend including a good sizing ladder such as the 1 Kb Plus ladder (Invitrogen).

      For successful TILLING, we need a robust product. Primers that do not amplify well at this step will be problematic in TILLING because the amount of product will not be sufficient for TILLING and the gels will be difficult to interpret. Ideally, you should see a single band on an agarose gel of the correct molecular weight for your target. The intensity of your PCR product (2 mL) should be at least 120 ng (make sure not to over saturate your image).

4. Sequence analysis

     In order to sequence the PCR products we clean up the products using Shrimp Alkaline Phosphatate (SAP) and Exonuclease I to deactivate the incorporated nucleotides and primers. We also add DMSO to our sequencing reactions. However, if the PCR was done with TMSO, we have found that the TMSO in the PCR sample is sufficient and add water in place of the DMSO. We encourage you to sequence the PCR product from both directions. Your sequence traces should be clean with a single strong peak at each base position. Below is a list of things that we have observed in our sequence analysis and what they tend to correlate with in TILLING.

   a) Weak sequencing reactions.

     If the sequencing reaction is consistently very weak, the TILLING gels also tend to be weak and we have a difficult time obtaining good sequence from the individual harboring the mutation.

   b) Heterozygous bases.

     A few heterozygous positions in your target are not a problem particularly if they are in an intron. We have observed that some bases in introns are naturally heterozygous. If these hets are in the exon, you may want to determine if you are working with a duplicated gene. If your sequence has many heterozygous positions (> 10), you should re-evaluate your gene target. The CelI step in TILLING assay will recognize these hets and cleave them. This results in background bands on the gel that may obscure real mutations.

   c) Run of a single nucleotide.

     If you have a run (> 8 bases) of a single nucleotide you will often see two traces, one of them shifted by a base. These targets are not optimal for TILLING because when we sequence verify the mutations, we will have a difficult time interpreting the sequence and confirming mutations.

   d) PCR product not the expected size.

     In our Primer Pre-Screens we have had cases where the B73/W22 gDNA sequence does not align perfectly with the user submitted sequence. Usually this discrepancy is due to introns that the user did not know about. As long as the PCR product is 1.5 kb or less this is not a problem (1.5 kb is our upper limit for TILLING PCR). You should, however, go back and re-CODDLe your gene with the correct gDNA sequence before submitting your order.

5. Placing the TILLING order

     Once you are satisfied with your level of testing, return to your saved html file that was described in section 2.1. Click the "order TILLING of this region" link and fill out the order form.