Gene Synthesis

Building up gene-length DNAs from single bases

Gene Synthesis

(aka DNA total synthesis)

Trends Biotechnol. 2009 Feb;27(2):63-72 PMID: 19111926

Current metrics

	IDT single-sequence oligonucleotide synthesis, 25 nmole scale, 2026	Twist Clonal Genes, 2026
Size	15-60 bases at this scale	0.3 - 5.0 kb insert
Price	$0.47 per base, US list	From 9¢ per bp, plus a cloning fee of $35 and up per construct
Yield	10nmol	1 ug - 1 mg
Turnaround time	Over 90% of orders ship in 24 hrs	10 business days
State	Chemically pure, sequence non-clonal, single-stranded	NGS-verified double-stranded plasmid clone

Vendor list prices, September 2026

Oligonucleotide Synthesis

Phosphoramidite oligonucleotide synthesis

A 35-mer primer runs about $5, and it is in hand the next day
Over 60 bases the order moves up a scale, and the price per base roughly doubles
Past about 100 it is a different product altogether — several vendors sell one, all of them much more expensive
They are synthesized 3’ to 5’, opposite that of polymerases
Non-clonal: deletions and truncations increase towards the 5’ end

Ligase Chain Assembly

Example: Gene synthesis oligo design

A sequence editor window showing the lambda repressor sequence, with restriction sites highlighted and the whole insert selected

Construct the complete sequence to be synthesized, including restriction sites and 5’ tails
Copy to the clipboard

Download the annotated file

Cut both strands, half an oligo out of step

Paste the complete cassette

Oligo length

nothing pasted yet

C6-Tools does this job too, inside the spreadsheet suite you get later in the course

LCA example question

Design a set of oligos to synthesize the amilGFP gene as a BioBricks part using Ligase Chain Assembly

amilGFP ORF:

ATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGC
AGTGTCAATGGCCATGAATTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGG
AAACAGATGTCCGAATTAGTGATCATCAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTT
GACATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCAGAC
ATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTA
TTTGAGGATGGAGCAGTTGCTACAGCCAGCTGGAACATTCGACTCGAAGGAAATTGCTTC
ATCCACAAATCCATCTTTCATGGCGTAAACTTTCCCGCTGATGGACCCGTAATGAAAAAG
AAGACCATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGGTGCTAAGA
GGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCAC
TCCACTTACAAAACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAA
ATTGTGAGGACCGACCTTGGCCAAAGTGCAAAAGGCTTTACAGTCAAGCTGGAAGCACAT
GCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAA

1. Put the BioBrick ends on the ORF — they are the blue stretches

ccattgaattcgcggccgcttctagATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGCAGTGTCAATGGCCATGAATTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGGAAACAGATGTCCGAATTAGTGATCATCAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTTGACATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCAGACATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTATTTGAGGATGGAGCAGTTGCTACAGCCAGCTGGAACATTCGACTCGAAGGAAATTGCTTCATCCACAAATCCATCTTTCATGGCGTAAACTTTCCCGCTGATGGACCCGTAATGAAAAAGAAGACCATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGGTGCTAAGAGGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCACTCCACTTACAAAACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAAATTGTGAGGACCGACCTTGGCCAAAGTGCAAAAGGCTTTACAGTCAAGCTGGAAGCACATGCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAAactagtagcggccgctgcagacsgc

2. Cut both strands, 50 bases at a time

PCA Assembly

(Polymerase chain assembly)

PCA Assembly

(Polymerase chain assembly)

Start with overlapping 50-60bp Oligos
Combine in a thermostable polymerase reaction
Run a PCR-like program
Use “assembly product” as template for a conventional PCR with external oligos
Clone the product into a vector to generate a synthon

Cut once, and let the oligos overlap

Paste the complete cassette

nothing pasted yet

C6-Tools, the same pca() function, in the spreadsheet suite you get later in the course

PCA example question

Design a set of oligos to synthesize the amilGFP gene as a BioBricks part using Polymerase Chain Assembly

amilGFP ORF:

ATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGC
AGTGTCAATGGCCATGAATTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGG
AAACAGATGTCCGAATTAGTGATCATCAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTT
GACATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCAGAC
ATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTA
TTTGAGGATGGAGCAGTTGCTACAGCCAGCTGGAACATTCGACTCGAAGGAAATTGCTTC
ATCCACAAATCCATCTTTCATGGCGTAAACTTTCCCGCTGATGGACCCGTAATGAAAAAG
AAGACCATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGGTGCTAAGA
GGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCAC
TCCACTTACAAAACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAA
ATTGTGAGGACCGACCTTGGCCAAAGTGCAAAAGGCTTTACAGTCAAGCTGGAAGCACAT
GCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAA

1. The same cassette as the LCA question — BioBrick ends in blue

ccattgaattcgcggccgcttctagATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGCAGTGTCAATGGCCATGAATTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGGAAACAGATGTCCGAATTAGTGATCATCAAGCCTGCGGGAAAACCCCTTCCATTCTCCTTTGACATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCAGACATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTATTTGAGGATGGAGCAGTTGCTACAGCCAGCTGGAACATTCGACTCGAAGGAAATTGCTTCATCCACAAATCCATCTTTCATGGCGTAAACTTTCCCGCTGATGGACCCGTAATGAAAAAGAAGACCATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGGTGCTAAGAGGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCACTCCACTTACAAAACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAAATTGTGAGGACCGACCTTGGCCAAAGTGCAAAAGGCTTTACAGTCAAGCTGGAAGCACATGCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAAactagtagcggccgctgcagacsgc

2. Hand it to PCA

Why don’t we always do this?

In practice it’s slow (our designs took 6 months)
As a non-clonal fragment it only gets you to 3kb before you need to break into two steps, due to mutation rates; sequence-verified clonal genes go further (7kb at Twist, 15kb at Elegen, 50kb at Ansa)
Difficult to synthesize repetitive sequences, biased GC-content, secondary structure (promoters and terminators are problematic)

$$

Total number of bases for 2014’s 140L: 43517

Would cost about ~$6,000 for basic parts

Would cost ~$200,000 for all the composite parts

Twist Clonal Genes list price, September 2026