Why NCBI Tools are important for
breeding plants studies
genetically modified organism:
the impossibility of intergenic crosses caused by the genetic
incompatibility between two species related e.g. between
barley and wheat , make the process of integration of a gene
responsible of the expression of a trait with a big agronomic
interest so dificult
This is why genetic engineering has become a very important
tool to solve this big issue
• At the end of the process of transformation
we need to do a screening to reveal the
integration of the new gene in the recipient
organism .
How can we reveal the integration?
One of the most succesfull screening process is
the PCR!
How NCBI Tools can help us with our
PCR
• The Main factor to amplify the target gene is
the primer.
• So how can we design the suitable primer to
do the revealing experiment ??
Welcome to Pick primer
• Step 1: open the NCBI Home page.
• Step2: target gene sequence reaserch.
Step 3: research results
Step 4: The GenBank Format
Step 5: convert from GenBank format To Fasta Format
GenBank
format
FASTA
Format
Step 6:click on the Tab"pick primers"
Step 7:Enter accession or FASTA sequence and
the primer parametres
Step 8 : Primer Pair Specificity Checking
Parameters
Step 9: Click
Sequences of the first primer Pair
• Things you can do to maximize the chance of finding primers
specific for your template.
• Use refseq accession or GI (rather than the raw DNA sequence) as
template whenever possible. In addition, make sure you are using
the latest version of a refseq entry (i.e., the accession with the
highest version number or simply just use the accession without the
version number as you will automatically get the latest version).
• Even if you are only interested in part of the sequence (for example,
a region on chromosome), you should still use the accession or GI
but you do need to specify the range (use forward primer "From"
field for your sequence start position and reverse primer "To" field
for your sequence stop position). The reason is that an accession or
GI carries accurate information about its identity which allows
primer-blast to better distinguish between intended template and
off-targets.
•
Choose a non-redundant database (such as refseq_rna or genome database). The nr
database contains redundant entries which can interfere with the process of finding
specific primers.
•
Specify an organism for database search if you are only amplifying DNA from a specific
organism. Searching all organisms will be much slower and off-target priming from
other organisms are irrelevant.
•
Exclude certain sequences such as predicted transcripts from database search if you are
not concerned about these.
•
Lower the primer specificity requirements if you are only concerned with targets that
have perfect or nearly perfect matches to your primers...By default, primer-blast uses
stringent parameters that can detect targets having significant number of mismatches
to primers (in addition to perfectly matched targets). See Primer specificity stringency
under Primer Pair Specificity Checking Parameters for more details.
•
Loosen other restrictions if necessary. Sometimes specific primers may have been
excluded due to location restrictions such as exon junctions, SNPs, low complexity and
repeat sequence filtering.