Step3: Design Gene-Specific Primers
Go to /Blast.cgi
Scroll down to find the Specialized BLAST, click Primer BLAST
NCBI Primer-BLAST
1. Enter accession number or FASTA sequence (mandatory)
• This program does NOT:
– Check for repeat sequences within the amplicon which may lead to stutter – Check for pseudogenes
Design Gene-Specific Primers with NCBI Primer-BLAST
• Homology and/or transcript variants • SNP • Intron/exon boundaries are known
The shotgun approach is an efficient method to use when little or no genome information is available for the organism of interest.
NCBI web or directly go to /gene/
1. Choose the
Gene database
2. Enter gene
name or accession number
3. Click Search
Get the alias(es) and a summary
• Do not use an accession number for genomic sequences
2. Ensure the following for each accession number that is chosen:
• Correct gene is chosen
– – – – Multiple names and aliases Different genes can have similar names Species of interest If non-RefSeq accession number is used Verify that the sequence contains only the letters A, T, G, C or N
– Choosing the gene and accession number
• Use only mRNA accession numbers
– Amplicon length and space between peaks
• Design using NCBI Primer-BLAST
– Amplicon lengths – Design primers to detect all transcript variants (if not specified) or a unique transcript variant (if specified) – Design primers to avoid amplifying transcribed pseudogenes – Design intron-spanning primers when possible – Exclude SNPs from primers
Pre-Design Considerations-Continue
• Gene Information • Intron and Exon Information • Transcript Variants Information • Pseudogenes Information
Step1: Getting Gene Information
Advanced Primer Design Workflow
Obtain mRNA accession numbers or use gene sequence Enter primer name, fragment size, gene name into multiplex TDF file template
• Little or no homology, pseudogene or transcript variant information • No SNP information • No intron/exon boundaries
Strategic Approach
Accession Numbers
Pre-design considerations
NCBI Primer-BLAST design
Order primers with universal tags and evaluate primers in vitro
Strategic Approach
• Pre-design considerations
(without universal tags)
Design primers using Primer BLAST
Pre-Design Considerations
1. Choosing an Accession Number
• A single gene can be represented in the NCBI database by multiple accession numbers – mRNA » Always use reference sequence (RefSeq) (NM_XXXXXX) from the Database » Use caution with other mRNA accession numbers - partial sequences - mutations - ESTs
Enter the accession # here
Select “reference only” database Click “Begin Search”
Click View Report to view gene information
Click “View Report”
Report view page
• This program will accomplish the following when the parameters are set properly:
– Check for specificity both within a species and between species (must add species) – Include or exclude transcript variants – Exclude SNPs from the primer binding sites – Create intron-spanning primers and/or exon-junction primers – Allows for design to specific regions within the gene or using a pre-designed primer sequences – Avoids low complexity primer binding regions – Note: Generally the more restrictions that are placed on the primer selection program, the fewer primers binding sites will be available
Step 2: Checking for homology and pseudogenes
Go to /Blast.cgi
Perform a species-specific query
Blast human genome if it is a human gene
– 142 – 387 nt with universal tags – Start with small fragment size and work toward larger fragment size
Note: For FFPE samples, design amplicons between 105-160 nt (without universal tags), the recommended total number of fragments in a panel is twenty or less.
GeXP Gene Expression Multiplex Design:
Using NCBI Primer-BLAST to design high performance, gene-specific primers for an XP-PCR multiplex
Updated: March 07, 2011
Check gene for transcript variants, pseudogenes and intron information: Entrez Gene (See Specific Design Considerations)
Determine the desired length of amplicon
NM_018255
Black hash marks indicate exon-exon boundaries
Only one hit, there’s no high homologous region or pseudogenes.
Click on the link to go to the map view and intron-exon information.