Designing Primers for PCR/Sanger Sequencing Using Ensembl and NCBI Primer-BLAST: Step-by-Step Guide

Designing primers to amplify specific genomic regions is a core task in molecular diagnostics. This guide explains how to design primers using the Ensembl Genome Browser and NCBI Primer-BLAST, with a real-world example: the SNV RET:c.320A>T on transcript ENST00000355710.8.

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Step 1: Open Ensembl and Search for the RET Gene

1. Visit the Ensembl Genome Browser.
2. In the search bar:
   • Select Human from the species dropdown.
   • Enter the gene name: RET.
3. Click on RET – Human Gene in the results.

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Step 2: Choose the Transcript of Interest

You’ll see a list of transcripts (splice isoforms) of the RET gene.

• Identify the one relevant to your clinical or experimental context.
• Most commonly, you’ll select the one labeled MANE Select.

What is MANE Select?
The MANE (Matched Annotation from NCBI and EMBL-EBI) Select transcript is a reference transcript agreed upon by both Ensembl and RefSeq. It represents the biologically most relevant transcript for clinical reporting.
You’ll find “MANE Select” written next to the transcript ID—for example:
ENST00000355710.8 (MANE Select)

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Step 3: Locate the Variant in the cDNA View

1. Click “Sequence” from the left-hand menu.
2. Choose the “cDNA” option to view the coding DNA sequence.
3. Search for your variant by scrolling or using Ctrl + F to find c.320A.

In this view:

• The top row shows complementary DNA (non-coding strand).
• The bottom row shows the coding DNA sequence (cDNA), which you’re interested in.

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Step 4: Identify the Variant in Genomic Context (Exon View)

To locate the exact genomic location of the SNV in the context of exons and introns:

1. First, copy ~20 nucleotides upstream and downstream of the SNV from the cDNA view.
   • This short flanking sequence helps you locate the SNV in the exon view.
2. Now click “Exons” in the left panel.

1. Use Ctrl + F to search for the copied flanking sequence.
2. Important setting:
   • Make sure to check the box “Show full intronic sequence”.
   • This allows visualization of both exons and introns in the gene.

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Step 5: Extract the Target Sequence Around the SNV

1. Once you find the position of c.320A in the exon/intron sequence:
   • Copy ~100–200 base pairs upstream and downstream of the SNV.
   • You can include intronic sequences if the SNV lies near exon-intron boundaries.
   • If difficult to copy in one stretch, use Notepad/Word to arrange the sequences

This is your target sequence that will be submitted to Primer-BLAST.

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Step 6: Go to NCBI Primer-BLAST

1. Open NCBI Primer-BLAST.
2. Paste the copied sequence in the “PCR Template” box.

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Step 7: Configure Primer-BLAST Parameters

1. Define primer binding site ranges:
   • Example: If the SNV lies at position 150 in a 300 bp sequence:
     • Set Forward Primer range: 1–100
     • Set Reverse Primer range: 200–300
   • This ensures that primers flank the SNV and do not overlap it.
2. Database:
   • Choose RefSeq representative genomes.
3. Organism:
   • Select Homo sapiens.
4. Optional primer settings:
   • Product size range: 200–400 bp
   • Primer Tm: 58–62°C
   • Max Tm difference: 2°C
   • You can leave the other default settings unchanged.

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Step 8: Review Primer-BLAST Results

1. Click “Get Primers”.
2. Primer-BLAST will return a list of primer pairs.
3. Select primer pairs that:
   • Flank the SNV (ideally with at least 30 bp gap between primer end and the SNV).
   • Have good parameters:
     • GC content: 40–60%
     • Melting temperature (Tm): ~60°C
     • Low self-complementarity and 3′ complementarity
     • Amplicon size: 200–400 bp

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Step 9: Final Specificity Check (Optional)

To be extra sure, BLAST each primer individually using NCBI BLAST:

• Confirm that each primer binds only to your intended target.
• Ensure that no off-target or pseudogene regions are amplified.

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Summary Table

Step	Action
1	Open Ensembl and search RET
2	Select transcript with “MANE Select” label
3	Find c.320A in cDNA view
4	Use flanking bases to locate SNV in exon view
5	Copy 100–200 bp surrounding SNV
6	Paste into NCBI Primer-BLAST
7	Define binding ranges, select Homo sapiens
8	Choose best primer pair flanking SNV
9	Confirm specificity via BLAST (optional)

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Conclusion

With just Ensembl and Primer-BLAST, you can reliably design PCR primers tailored to your variant of interest. The key steps are:

• Identifying your variant accurately,
• Extracting a clean genomic context,
• Flanking the SNV appropriately,
• And choosing primer pairs with ideal melting properties and specificity.

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