Primer Assembly Protocol¶

What is Primer Assembly?¶

Primer assembly (also known as oligonucleotide assembly or gene synthesis by assembly) is a molecular biology technique used to construct longer double-stranded DNA combining shorter synthetic oligonucleotides (primers) using PCR

Definition¶

Primer assembly is the process of: 1. Designing overlapping oligonucleotide sequences that span the target DNA/RNA sequence 2. Synthesizing these short DNA fragments (typically 20-60 nucleotides each) 3. Annealing complementary overlapping regions between adjacent oligonucleotides 4. Extending the annealed fragments using DNA polymerase to create full-length double-stranded DNA 5. Amplifying the assembled product using PCR

Why Do We Use Primer Assembly?¶

Primer assembly is used for several important reasons:

1. Cost-Effective Gene Construction¶

Synthesizing long DNA sequences (>200 bp) directly is expensive
Short oligonucleotides (20-60 bp) are much cheaper to synthesize
Assembly allows construction of genes, promoters, or regulatory elements at a fraction of the cost

2. Custom Sequence Design¶

Enables creation of sequences that don't exist in nature
Allows introduction of specific mutations, deletions, or insertions
Permits design of optimized sequences (codon optimization, regulatory elements)

3. Rapid Prototyping¶

Faster than traditional cloning methods for new constructs
Can test multiple sequence variants quickly
Enables iterative design and testing cycles

4. Precision and Control¶

Exact control over every nucleotide in the sequence
Can introduce specific modifications (mutations, tags, linkers) precisely
Avoids issues with restriction sites or unwanted sequences from traditional cloning

5. Building Complex Constructs¶

Assemble multiple functional elements (promoters, coding sequences, terminators)
Create libraries of related sequences with systematic variations
Construct chimeric sequences combining elements from different sources

6. RNA Structure Studies¶

For RNA research, primer assembly allows construction of RNA sequences with specific structural features
Can introduce modified nucleotides or specific sequences for structure-function studies
Enables creation of RNA libraries for high-throughput screening

When to Use Primer Assembly¶

Use primer assembly when: - Constructing sequences longer than ~200 bp - Need precise control over sequence - Creating sequences that don't exist naturally - Building multiple related variants - Cost is a consideration (cheaper than full gene synthesis) - Need to introduce specific mutations or modifications

Consider alternatives when: - Sequence already exists in a plasmid (use PCR or restriction cloning) - Very short sequences (<100 bp) - direct synthesis may be simpler - Need very long sequences (>3 kb) - may need hierarchical assembly or commercial synthesis

Materials¶

Reagents¶

Oligonucleotides: Overlapping primers designed for assembly (typically 20-60 nucleotides each, 15-20 bp overlaps)
DNA Polymerase: High-fidelity polymerase (e.g., Q5, Phusion, or Pfu)
dNTPs: Deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
PCR Buffer: 10x buffer appropriate for your polymerase
Nuclease-free water: For dilutions and reactions
Agarose: For gel electrophoresis
DNA ladder: Molecular weight marker
Gel loading dye: 6x loading buffer
Ethidium bromide or alternative DNA stain: For visualization

Equipment¶

Thermal cycler: For PCR reactions
Gel electrophoresis apparatus: For analyzing products
UV transilluminator or gel imaging system: For visualizing DNA
Micropipettes: P10, P20, P200, P1000
PCR tubes or 96-well plates: For reactions
Ice bucket: For keeping reagents cold
Centrifuge: For spinning down reactions

Software/Tools¶

Primer design software:
NUPACK (for secondary structure prediction)
Primer3 (for primer design)
Custom scripts for overlap design
Sequence analysis tools:
BLAST (for checking sequences)
Sequence alignment tools

Protocol¶

Step 1: Design Overlapping Primers¶

1.1 Determine Target Sequence¶

Define the complete sequence you want to assemble
Include any modifications, tags, or mutations
Verify sequence is correct (check for errors, unwanted sequences)

1.2 Design Overlapping Oligonucleotides¶

Design parameters: - Length: 40-60 nucleotides per oligonucleotide (optimal) - Overlap: 15-20 base pairs between adjacent oligonucleotides - Melting temperature (Tm): Aim for 55-65°C for overlaps - Avoid: Secondary structures, repetitive sequences, homopolymers

Example design:

Target: 200 bp sequence

Oligo 1:  [1-50]     (50 bp)
Oligo 2:  [35-85]    (50 bp, overlaps oligo 1 by 15 bp)
Oligo 3:  [70-120]   (50 bp, overlaps oligo 2 by 15 bp)
Oligo 4:  [105-155]  (50 bp, overlaps oligo 3 by 15 bp)
Oligo 5:  [140-190]  (50 bp, overlaps oligo 4 by 15 bp)
Oligo 6:  [175-200]  (25 bp, overlaps oligo 5 by 15 bp)

1.3 Add Outer Primers¶

Design forward and reverse primers that bind to the 5' and 3' ends
These will be used for final PCR amplification
Include restriction sites, tags, or other modifications if needed

1.4 Verify Primer Design¶

Check for secondary structures using NUPACK or similar
Verify no primer-dimers will form
BLAST primers to check for off-target binding
Calculate melting temperatures

Step 2: Order Oligonucleotides¶

Order from commercial supplier (IDT, Sigma, etc.)
Specify synthesis scale (typically 25 nmol is sufficient)
Request standard desalting purification (usually sufficient)
Verify sequences when received

Step 3: Prepare Oligonucleotide Stocks¶

3.1 Resuspend Oligonucleotides¶

# Typical resuspension
# For 25 nmol scale, add 250 μL nuclease-free water
# Final concentration: ~100 μM

# Calculate volume needed:
# Volume (μL) = (nmoles × 10^6) / (desired concentration in μM)

3.2 Dilute to Working Concentration¶

Create 10 μM working stocks for assembly reactions
Store at -20°C for long-term storage
Keep on ice when working

Step 4: Assembly PCR¶

4.1 Set Up Assembly Reaction¶

Reaction setup (50 μL total volume):

# Typical reaction mix:
# - 1-2 μL of each oligonucleotide (10 μM stock)
# - 1x PCR buffer
# - 200 μM each dNTP
# - 0.02 U/μL DNA polymerase
# - Nuclease-free water to 50 μL

# Example for 6 oligonucleotides:
Component              Volume
─────────────────────────────────
Oligo 1 (10 μM)        1 μL
Oligo 2 (10 μM)        1 μL
Oligo 3 (10 μM)        1 μL
Oligo 4 (10 μM)        1 μL
Oligo 5 (10 μM)        1 μL
Oligo 6 (10 μM)        1 μL
10x PCR Buffer         5 μL
dNTPs (10 mM each)     1 μL
Polymerase             0.1 μL (or per manufacturer)
Water                  38.9 μL
─────────────────────────────────
Total                  50 μL

4.2 Thermal Cycling Program¶

Typical program:

Step 1: Initial denaturation
  98°C for 30 seconds

Step 2: Annealing and extension (5-10 cycles)
  98°C for 10 seconds    (denaturation)
  55-60°C for 30 seconds (annealing - adjust based on Tm)
  72°C for 30-60 seconds (extension - 1 min per kb)

Step 3: Final extension
  72°C for 2-5 minutes

Step 4: Hold
  4°C hold

Notes: - Annealing temperature should be 5-10°C below lowest overlap Tm - Extension time: ~30 seconds per 500 bp - Number of cycles: Start with 5-10, can increase if needed

Step 5: Amplification PCR¶

5.1 Set Up Amplification Reaction¶

Use outer primers to amplify the assembled product:

Component              Volume
─────────────────────────────────
Assembly product       1-5 μL (from Step 4)
Forward primer (10 μM)  1 μL
Reverse primer (10 μM)  1 μL
10x PCR Buffer          5 μL
dNTPs (10 mM each)      1 μL
Polymerase             0.1 μL
Water                  to 50 μL
─────────────────────────────────
Total                  50 μL

5.2 Thermal Cycling Program¶

Step 1: Initial denaturation
  98°C for 30 seconds

Step 2: Amplification (25-35 cycles)
  98°C for 10 seconds
  55-65°C for 20 seconds (primer Tm - 5°C)
  72°C for 30-60 seconds (1 min per kb)

Step 3: Final extension
  72°C for 2-5 minutes

Step 4: Hold
  4°C hold

Step 6: Analyze Products¶

6.1 Gel Electrophoresis¶

Run 5-10 μL of PCR product on 1-2% agarose gel
Include DNA ladder
Run at appropriate voltage (5-10 V/cm)
Visualize under UV light

6.2 Expected Results¶

Assembly PCR: May show faint or multiple bands
Amplification PCR: Should show single band at expected size
If multiple bands: Optimize conditions or gel-purify correct band

Step 7: Purify and Verify¶

7.1 Purify PCR Product¶

Use PCR purification kit or gel extraction
Elute in appropriate volume (typically 30-50 μL)
Quantify using Nanodrop or similar

7.2 Verify Sequence¶

Option 1: Sanger sequencing
Send purified product for sequencing
Verify sequence matches design
Option 2: Restriction digest
If designed with restriction sites, digest and check on gel
Option 3: Clone and sequence
Clone into vector, pick colonies, sequence

Troubleshooting¶

Issue	Possible Causes	Solutions
No product in assembly PCR	• Oligonucleotides not annealing • Too few cycles • Wrong annealing temperature	• Check primer design and overlaps • Increase cycles to 10-15 • Lower annealing temperature by 2-5°C
Multiple bands in amplification	• Non-specific amplification • Primer dimers • Contamination	• Increase annealing temperature • Redesign primers • Use touchdown PCR • Gel-purify correct band
Product shorter than expected	• Incomplete assembly • Secondary structures blocking extension	• Increase extension time • Add DMSO (2-5%) or betaine • Redesign primers to avoid secondary structures
Product longer than expected	• Primer dimers • Non-specific amplification	• Increase annealing temperature • Redesign primers • Use hot-start polymerase
Low yield	• Too few cycles • Suboptimal conditions • Degraded reagents	• Increase cycles • Optimize temperature • Use fresh reagents
Sequence errors	• Synthesis errors in oligonucleotides • Polymerase errors	• Order from reputable supplier • Use high-fidelity polymerase • Sequence multiple clones

Best Practices¶

Design¶

Verify sequences carefully before ordering oligonucleotides
Check for secondary structures that might interfere with assembly
Design overlaps with appropriate Tm (55-65°C)
Avoid repetitive sequences or homopolymers in overlap regions

Experimental¶

Use high-fidelity polymerase to minimize errors
Keep oligonucleotides on ice when working
Use nuclease-free water and clean pipettes
Include negative controls (no template, no polymerase)
Optimize conditions for your specific sequence

Quality Control¶

Always sequence verify the final product
Check multiple clones if cloning (at least 2-3)
Document all modifications and deviations from protocol
Keep records of primer sequences and conditions used

Expected Results¶

Successful Assembly¶

Single band of correct size on gel
Sequence matches design (100% identity)
High yield (>100 ng/μL after purification)
Ready for downstream applications (cloning, etc.)

Typical Yields¶

Assembly PCR: Variable, often low yield
Amplification PCR: 50-200 ng/μL typical
After purification: 20-100 ng/μL

Time Required¶

Primer design: 1-2 hours
Oligonucleotide ordering: 1-3 days (shipping)
Assembly and amplification: 1 day
Verification: 1-3 days (sequencing)
Total: ~1 week (depending on sequencing turnaround)

Applications in the Lab¶

Primer assembly is commonly used for:

RNA Construct Design: Creating RNA sequences with specific structural features
Mutagenesis: Introducing specific mutations for structure-function studies
Library Construction: Building libraries of related sequences
Chimeric Constructs: Combining elements from different RNAs
Optimization: Creating codon-optimized or structure-optimized sequences

References¶

Gibson et al. (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nature Methods.
Stemmer et al. (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides." Gene.

Last updated: December 18, 2025