Seeking to learn the steps for using the BLAST tool in NCBI for designing primers for make a PCR a specific SNP.
you can design primer manually by choosing forward primer and check the %GC and Tm by using online calculator
https://www.facebook.com/groups/226548717538374/227886520737927/?notif_t=group_comment_reply
and reverse primer choose your primer and by using the like below choose reverse complementary and also check %GC and Tm.
http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html
I hope it will be helpful
Alternatively, you can use the position of interest , then paste it on UCSC and use Primer 3 software to find primers , then you need to validate these primers in the Insilico PCR .
Finding primer pairs to amplify areas adjacent to SNP is a rather tricky operation as you need to avoid some genomic segments!!!
Good Luck
Just download the sequence of your target gene from a reliable database such as NCBI or Ensemble. Save the FASTA sequence and try to make your own primers by choosing sequences and checking their properties on oligocalc or NCBI primer designing tool. Very briefly their Tm should be around 50-60 degrees with GC content not more than 60 % and they should not form hair pin neither should they form primer dimers.
How to: Design PCR primers and check them for specificity
ONE OR MORE PRIMER SEQUENCES
1. Go to the Primer BLAST submission form.
2. Enter one or both primer sequences in the Primer Parameters section of the form. If only one primer is available, a template sequence is also required. See "A Target Template Sequence..." below.
3. In the Primer Pair Specificity Checking Parameters section, select the appropriate source Organism and the smallest Database that is likely to contain the target sequence. These settings give the most precise results. For broadest coverage, choose the nr database and do not specify an organism.
4. Click the "Get Primers" button to submit the search and retrieve template and specificity information.
A TARGET TEMPLATE SEQUENCE OR ACCESSION NUMBER
1. Go to the Primer BLAST submission form.
2. Enter the target sequence in FASTA format or an accession number of an NCBI nucleotide sequence in the PCR Template section of the form. If the NCBI mRNA reference sequence accession number is used, the tool will automatically design primers that are specific to that splice variant.
3. If one or both primer sequences are to be used in the search, enter these in the Primer Parameters section of the form. Primer BLAST performs only a specificity check when a target template and both primers are provided.
4. In the Primer Pair Specificity Checking Parameters section, select the appropriate source Organism and the smallest Database that is likely to contain the target sequence. These settings give the most precise results. For broadest coverage, choose the nr database and do not specify an organism.
5. Click the "Get Primers" button to submit the search and retrieve specific primer pairs.
(Ref. https://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers)
May be this will helpful for your research. All the best.
- Badges
- Science method