Tutorial: Designing PCR primers to amplify a gene from genomic DNA
March 22, 2018
|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
LepR3 [Text Word] AND Brassica napus [ORGN]
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 most recent release of the genome annotation tells us that this gene is found on Chromosome A10 (ie. in the A genome of B. napus.)
you mouse-over the gene, represented by the green
line with arrows, you'll see that the gene is annotated as
spanning nucleotides 17,537,985..17,541,448. Note that the view is
shown with respect to the orientation of the LepR3 gene,
which is encoded on the reverse strand. Therefore, the
coordinates in the genome viewer go from high numbers to
low numbers, going left to right.
|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. a 3464 bp gene plus 1000 bases
upstream and 500 bases downstream, rounded to 5000).
|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 17,537,500 ).
|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
5 and 6 are shown.
that the Accession number is the same as the scaffold, and
the region shown in this file is the 5294 bp fragment
going from 17537355 to 17542648
. 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.