---+ Sequencing/Genotyping Primer Design %GREEN% This protocol is specifically for designing primers to PCR amplify a target region of interest from a genome and re-sequence it using the Sanger method. %ENDCOLOR% ---++ 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 scenarios.) [[http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=413997&INPUT_SEQUENCE=NC_012967.1&log$=seqview_list_primer][NCBI Primer Design for REL606 %ICON{"external"}%]] *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 [[http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi][blank form %ICON{"external"}%]] and fill out the sequence and organism fields. ---++ General Notes * Use a high-fidelity polymerase for all PCR reactions that will be re-sequenced. * Design all PCR products to be 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. * Sanger sequencing runs provide useful sequence starting at around 50 bases past the end of the primer and extending to approximately 600–800 bases past the end of the primer. ---++ 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. ---++ Strategy for any mutation in a gene * Include sequences upstream starting 100 bp before this gene or the transcription start site of this gene. * Include > 50 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. ---++ Ordering primers Follow the instructions at http://barricklab.org/twiki/bin/view/Lab/ProtocolsOrderingPrimers.
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Topic revision: r6 - 2011-06-06 - JeffreyBarrick