Sequencing/Genotyping Primer Design

This protocol is specifically for designing primers to PCR amplify a target region of interest from a genome and re-sequencing it using the Sanger method.

NCBI Primer Design Tool

Usually, we are interested in checking for mutations in Escherichia coli strains derived from REL606. While it's possible to design primers using a variety of tools, or even by eye, the NCBI website has a handy and free utility that we'll use in this protocol. (See additional instructions below on how to use this tool for different design scenarios.)

NCBI Primer Design for REL606

Variant: The link above automatically fills in certain fields with values specific to REL606. If you are studying another genome, you'll want to use a blank form and fill in the "PCR Template" box, and also the "organism" in the "Primer Pair Specificity Checking Parameters" section.

General Notes

• Use a high-fidelity polymerase for all PCR reactions that will be re-sequenced.
• Design primers for PCR to amplify DNA products that are 500–3,000 bp if possible.
  • The limit on the short side ensures that they are easily resolvable and sized in a 1% agarose gel.
  • The limit on the long side ensures that the product will be readily amplified under standard PCR conditions.
• Design primers for Sanger sequencing 50–600 bp upstream of the region of interest.
  • This region of the trace reliably produces usable sequence.
• Always perform control PCR/sequencing reactions with the ancestral strain.
  • This is a positive control for PCR with the primers you have designed working
  • Sequencing this ensures that the ancestor did not have this mutation.
• Always perform control PCR reactions with no template (a "blank").
  • This ensures that product is not due to amplifying contaminating template.

Strategy for a single mutation

Searching for Primers

The product of the PCR process should include at least 100bp on either end of the mutation of interest. For example, if this mutation was a deletion that ranged from approximately 1,000,000 to 1,001,000, then the region 999,900 to 1,001,100 should not be excluded when designing primers. To do this, enter ranges for the forward and reverse primer which do not cover this region. A decent range to begin with is 100bp. For example, if the region between 999,900 and 1,001,100 was to be copied, then a good initial search might be:

Forward Primer: 999,800 to 999,900

Reverse Primer: 1,001,100 to 1,001,200

Larger ranges result in longer search times, but tend to find more primer combinations. It will often be necessary to extend the ranges far outside the ends of the sequence of interest in order to find compatible primers. Generally speaking, it is better to have the primers too far away than too close to the sequence of interest. However, it is best for the sequence of interest to be centered on the product. As long as the final product is less that 2500-3000bp, there is no major problem with wide sequences. Note that even the link from this website puts the product size limit to 1000bp. If a larger product is needed, this value must be manually changed or you will get no matches.

Once several primers have been found, analyze the candidates. If multiple primer combinations fit, as they often will, selecting the first one is quite acceptable. The first thing to check for is if the primer attaches to any sites outside of the intended target. Fortunately, the website states in bold when this is the case, and any primers which do bind outside of the intended region should be disregarded. Next, primers with multiple binding sites within the intended region should also be disregarded. Only primers which have only 1 location for the forward primer and 1 location for the reverse primer to bind should be taken into consideration.

• For point mutations (SNPs) and insertions and deletions (indels) < 100 bp:
  Design two primers ~200-350 bp upstream and ~200-350 bp downstream of each mutation to amplify a 400-700 bp fragment. Sanger sequence from only one end. One reaction per template.

• For new insertion-sequence (IS) insertions:
  Design primers ~200-350 bp upstream and downstream of ends of mutation to amplify product. Do control reactions with REL606 to verify the expected size change. Sequence from both sides for IS-insertions so that we can determine the new IS orientation and new junction sequences.

• For large deletions (> 100 bp):
  Design primers ~200-350 bp upstream and downstream of ends of mutation to amplify a product for the deletion. If the product for the non-deleted ancestral genome is too large to reliably amplify (> 5 kb), then order an additional primer within the deleted interval that can amplify a 400-700 bp fragment with the original forward primer. Always do control reactions with REL606 to verify that a band appears in it for just one primer pair (the non-deleted specific pair) and that a band appears for the evolved genomes with just one pair (the deletion specific pair). Sequence the deletion from just one end. Be sure to design primer pairs such that the products of each PCR differ by >200 bp in length, so that you can tell them apart on the gel!

• For rearrangements, amplifications, and inversions:
  Design primers ~200-350 bp upstream and downstream of new junctions. PCR across the junction and Sanger sequence to verify the exact junction site. Be careful to NOT design primers in repetitive regions (such as IS elements, tRNA genes, rRNA genes, etc.) that could give confusing amplification products!

Strategy for general primers to test for any mutation in a gene

1. Look up the gene's start and end coordinates in REL606 using the Genbank file.
2. Search for the gene's page in the Ecocyc database.
   • Scroll to the bottom of the "Genes" page to see transcriptional units (TUs) that contain the gene (Gene Local Context).
3. If the gene is in the middle of a transcriptional unit, then use the start and end coordinates of the gene to design primers as indicated below.
4. If the gene is at the beginning of a transcriptional unit, then design primers that amplify a region that includes the transcription start site and upstream regulatory DNA binding sites.
   • Find the appropriate size by clicking on the picture of the TU with the furthest upstream promoter.
   • On the TU page, scroll to where you can find out how many bp it is upstream of the gene's start coordinate (Position relative to start of first gene).
5. Include sequences upstream starting >100 bp before this gene or the transcription start site of this gene.
6. Include >100 bp downstream of the end of the gene.

If you are ordering primers that tile a gene for sequencing, be conservative and assume that you will get usable Sanger sequence data extending from 50–550 bases past the end of each sequencing primer.

Primer Design Specifics

• Details for good primer design using the REL606 NIH Link:
  1. Change PCR Product size as needed. This means changing the minimum and maximum size to give a window that makes sense for your application. Examples:
     • If you need at least 100 bp of homology on either side of a mutation, the minimum size needs to be 200.
     • If you are amplifying across a 5kb deletion, the minimum size needs to be 5kb.
  2. The primer melting temperatures should always be changed as follows: Min: 60, Opt: 65, Max: 72
     • Optimal can be increased up to the Max.
     • Max should not increase above elongation temperature of your polymerase. 2 step PCR can be preformed if melting temperature is equal to elongation temperature.
  3. Range for primers should be at least 50-100 bp in length for both forward and reverse primers.

• Details for good primer design using jPCR http://primerdigital.com/tools/pcr.html. Believed to be the same instructions as for fastPCR if on windows http://primerdigital.com/fastpcr.html .
  1. With cursor in "Sequences" Tab at bottom of java window: File: Open file(s) into current tab
     • Select valid Fasta reference file
  2. Manually enter what you want your forward and reverse primer sequences to be in fasta format on the "Pre-designed primer (probe) list" tab. This is best done by copy pasting sequences from Genious or other genome browser.
  3. Select the "in silico PCR" tab at the top and click the green arrow
  4. Use the newly created "in silico PCR result" tab at the bottom to analyze your primers based on the criteria in the following section.

• Determining if the primers it picks for you are going to work:
  1. in silico PCR vs. blast.
     • At least 1 of these methods should be used for all primer pairs before ordering them.
     • in silico PCR is preformed by default by the NIH Primer design tool.
       • If there are multiple primer binding sites with the correct orientation as to give a PCR product, they will be listed as "Products on potentially unintended templates". If multiple products are listed with similar sizes, it will be impossible for you to distinguish the correct product from the unintended product. Therefore you should change the search range for 1 or both primers.
       • The NIH tool does not report mispriming sites of a single primer when it does not predict a product. This is often enough of a check, depending on downstream applications.
       • The jPCR section above has details for a combination in silico PCR and Blast
     • Blast is performed as part of the in silico PCR step in jPCR, and can be preformed separately via the NCBI blast website. Remember to change organisms if not using REL606.
     • Blast results should be evaluated based on mismatches and gaps at mispriming sites.
  2. The most important characteristic of determining good primers is Mispriming sites.
     • A mismatch on the final 3' base of each primer should not be able to elongate.
     • The more mismatches are present near the 3' end of the primer, the better the primer is.
     • Mispriming Tm greater than 40 is generally a bad sign. (Not given in NCBI Blast results)
     • In general the more mispriming sites there are, the worse the primer is.
  3. Tm of primers.
     • The closer the Tms are, the better.
  4. GC content.
     • Primers with very different GC content may not function well together.

• Troubleshooting for NIH design
  • No primers returned:
    1. Region for primers should be the first thing you increase if no viable primers are found.
  • low tm
    1. Under advanced options increase the max primer size from 25 to 32.
    2. If problem persists at a length of 32, increase to 35. If increased above 35, additional primer purification should be considered.

Ordering Primers

Follow the protocol for Ordering Primers.