Tutorial: Designing PCR primers to amplify a gene from genomic DNA
March 29, 2017
|go into tutorials directory
create a directory called primerblast
go into the primerblast directory
we'll need to use NCBI PrimerBlast, it's best to start by
finding the gene on the NCBI web site at https://www.ncbi.nlm.nih.gov.
Using the search panel at the top of the page, Set the
database to Gene, and type in the search term as shown:
The top part of the results are presented at right. Note that the formal annotation for this gene lists it as a receptor-like protein. This makes sense, since most plant disease resistance genes are Toll-like receptor protein kinases.
The genome annotation tells us that this gene is found on an unplaced scaffold. It should be noted that the publication for this gene places LepR3 on Chromosome 10A (ie. in the A genome of B. napus.) That suggests that although the genome assembly was unable to link this scaffold to a specific chromosme, this scaffold probably comes from Chromosome 10A.
|Primer-BLAST lists the accession number of
the genomic scaffold, and the coordinates of the selected
region are shown in the Forward primer and Reverse Primer
the Primer Parameters section, we set the Minimum PCR
product size to 5000 (ie. 299,000 - 294,000)
|Primer-BLAST chooses an Excluded region using
a starting point and a length. Therefore, type in
(No commas are allowed in large numbers such as 294,000).
|How does Primer-BLAST work?
The search for primers is essentially a 2-step process:
1. Use the Primer3 program to design candidate primer pairs for the target sequence. Almost all of the parameters to Primer-BLAST are actually parameters for Primer3.
2. Use MegaBLAST to search an NCBI database for matches to the primer. Any good matches to genes in the database other than the target sequence will cause candidate primers to be discarded. This step is critical to ensure that the final primer pairs will amplify the desired target, and no other sequences in the genome. By default, the RefSeq mRNA database is searched for unwanted matches.
you scroll down to the Detailed Primer Reports, you will
see the sequences of the primers, as well as other data on
the PCR primers. For comparison, results for Primer pairs
2 and 3 are shown.
that the Accession number is the same as the scaffold, and
the region shown in this file is the 5179 bp fragment
going from 293914 to 299092. Save the file using the Send
menu in the upper right corner, and choose Complete
record, File, and GenBank (full).
Click on Create File to save, and save as LepR3-PCR.gen.