PrimerBLAST 操作说明

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.
Pre-design considerations
NCBI Primer-BLAST design
Order primers with universal tags and evaluate primers in vitro
Strategic Approach
• Pre-design considerations
1. Enter accession number or FASTA sequence (mandatory)
Check gene for transcript variants, pseudogenes and intron information: Entrez Gene (See Specific Design Considerations)
Determine the desired length of amplicon
Enter the accession # here
Select “reference only” database Click “Begin Search”
Click View Report to view gene information
Click “View Report”
Report view page
Two Design Strategies
Use Accession Numbers or Proprietary Genes to create genespecific primers for amplicons 105-350 nucleotides in length
1 2
The strategic approach is recommended when a model organism is used and/or there is sufficient genomic data available in NCBI for PrimerBLAST to scan primers for:
– This allows for variation in migration to meet the minimum peak separation distance of 3 nucleotides.
•
Design amplicons between 105 – 350 nt (without universal tags)
• Homology and/or transcript variants • SNP • Intron/exon boundaries are known
Biblioteka Baidu
The shotgun approach is an efficient method to use when little or no genome information is available for the organism of interest.
(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
• Little or no homology, pseudogene or transcript variant information • No SNP information • No intron/exon boundaries
Strategic Approach
Accession Numbers
Example: NM_01825
View the transcripts and reference sequences
ELP2 contains a single transcript with 22 exons Each green bar represent an exon.
ELP2 located at the Chromosome 18
• 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
Step3: Design Gene-Specific Primers
Go to http://blast.ncbi.nlm.nih.gov/Blast.cgi
Scroll down to find the Specialized BLAST, click Primer BLAST
NCBI Primer-BLAST
Advanced Primer Design Workflow
Obtain mRNA accession numbers or use gene sequence Enter primer name, fragment size, gene name into multiplex TDF file template
Step 2: Checking for homology and pseudogenes
Go to http://blast.ncbi.nlm.nih.gov/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
NCBI web or directly go to http://www.ncbi.nlm.nih.gov/gene/
1. Choose the
Gene database
2. Enter gene
name or accession number
3. Click Search
Get the alias(es) and a summary
• 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
– 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
Amplicon Length
• Design amplicons such that each fragment is no less than 5 nucleotides apart from its nearest neighbor.
• 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
Pre-Design Considerations-Continue
• Gene Information • Intron and Exon Information • Transcript Variants Information • Pseudogenes Information
Step1: Getting Gene Information