Home › Immunology › Primer-Free Aptamer Selection Using A Random DNA Library
Immunology JoVE (Open Access) Citable · DOI

Primer-Free Aptamer Selection Using A Random DNA Library

DOI: 10.3791/2039-v
What you'll learn
  • ✓ Execute primer-free SELEX to eliminate fixed primer sequence interference
  • ✓ Prepare random DNA library fragments without flanking primer regions
  • ✓ Perform iterative selection and recovery of aptamers against target protein
  • ✓ Validate selected aptamers using sandwich binding assays
Protocol

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Difficulty
advanced
Total time
~5–7 days (including multiple selection rounds, cloning, and sequencing)
Biosafety
BSL-1

Steps

1
Understand primer-free aptamer selection rationale

Learn how fixed primer sequences in standard SELEX can cause false positives and negatives, and how primer-free methodology addresses this limitation.

▶ 00:12
2
Prepare primer-free library DNA fragments

Generate random DNA library fragments without flanking primer sequences to eliminate interference during selection rounds.

▶ 01:39
3
Prepare S100B-bound bead targets

Immobilize the target protein S100B on beads to create the selection substrate for aptamer binding.

▶ 03:26
4
Execute selection and aptamer recovery

Perform iterative rounds of selection by incubating library with S100B-beads, isolating bound aptamers, and recovering selected sequences.

▶ 04:55
5
Clone selected aptamers and sequence

Use TOPO cloning to insert recovered aptamer sequences into plasmids and perform DNA sequencing to identify selected clones.

▶ 06:40
6
Validate aptamers via sandwich binding assay

Perform binding assays to confirm selected aptamers specifically bind target protein and evaluate affinity.

▶ 07:17
💬 Comments coming soon