error-corrected dna synthesis peter carr mit media laboratory

Error-Corrected DNA Synthesis

Peter CarrMIT Media Laboratory

A C T G

DNA and a few handy tools

A C T G

Polymerase reads (copies) DNA

TA C T GA C T G T AA C T G T A C

CAGT GGTA

MutS sticks to mistakes

A C T G T A G

CAGT GGTA

T

CAGT

DNA comes in complementary strands

(A with T; G with C)

Uses for DNA On-Demand

basepairs

102 107105104103 106

genetic circuits genome rewrite

minimal lifesingle genes*IN

SERT: your fa

vorite

gene(s)

INSERT: y

our favo

rite gene(s)

Recoding E.coli: rE.coli(with Church & Isaacs)

Step2. >4000 changesremove rare Arg codingfree two more codons

Step 3. >70,000 changesswap Leu and Ser codonsorthogonal genetic codes!

Step1. >300 changesTAG stops become TAA stopsleaves one codon freeE. Coli

MG16554.6 MB

Current “price tag” >$3 million per genome

(and we want several of these)

Trends in de novo DNA synthesis

Carlson, R. (2003) The Pace and Proliferation of Biological Technologies

user designsDNA

DNA Fabrication Platform:Integration Road Map

oligomicroarraysynthesis

assembleDNA constructs

DNA errorcorrection

larger scaleassembly

clone

express/assay

sequence/QC

MOLECULESDATA DEVICE

DATADATA DEVICE (molecules)


inkjet-printed microarrays(e.g. Agilent)

maskless array synthesizer(e.g. Nimblegen)

High Density Oligonucleotide Microarrays

a massive feedstock of DNA building blocks

>1000x reduction in oligonucleotide costs

>105 oligos permicroarray

>5 megabasesof DNA information= =

Tian & Church (2004):~600 oligos 21 genes 15 kb construct

user designsDNA




DNA errorcorrection


clone

express/assay

sequence/QC





Polymerase Construction and Amplification (PCA)

Four parallel 500-nL reactors

Parallel microfluidic gene synthesis

PDMS1

PDMS2

PDMS3

Fabrication

Parallel microfluidic gene synthesis

GeneTotal size (nt)

Number of oligos

Construction Oligo size (nt)

Amplifying Primer sizes (nt)

alba 327 16 38 35, 32

hjc 390 16 48 25, 25

ble 461 16 47 31, 45

DsRed 733 26 50 25, 20

OR128-1 942 32 50 31, 37

GFP 993 42 42 29, 29

Kong et al. Nucleic Acids Research 35 2007

user designsDNA




DNA errorcorrection


clone

express/assay

sequence/QC



DNA errorcorrection

Error Correction for DNA

A C T G

A C T T

G C T G

CAGT

CAGT

AAGT

A C T G

CAGT

>109 copies in solution

probability of correlated errors is low

iteration (with strand re-partnering) makes more robust

(keep)

A C T G

CAGT

(edit)

(remove)

DNA Error Removal

bind MutS

removeMutS +

mismatch

(error-free DNA) Carr et al. NAR 32 (2004)

1 error per 104

base pairs

user designsDNA




DNA errorcorrection


clone

express/assay

sequence/QC





Hierarchical gene synthesis

103 – 104 base pairs

user designsDNA




DNA errorcorrection


clone

express/assay

sequence/QC



express/assay


Integrated gene and protein synthesis

Parallel synthesis of three genes followed by expression of fluorescent protein (dye simulation)

Synthetic EGFP observation by confocal fluorescence microscopy

Prof. Joseph Jacobson

David KongJason Park

Prof. Franco CerrinaProf. George ChurchDr. Shuguang Zhang

Funding: MIT Media LabCenter for Bits and Atoms (NSF)

Thanks to:

1 mm

Integrated gene and protein synthesis

oligos gene protein

45 nL gene synthesis reactors x3

12 nL protein synthesis reactors x3

Can we synthesize from oligos, in parallel, genes for multiple different proteins, express them, and assay their function?

DNA Error Correction Protocols

Hybridization-selection

Tian et al. Nature 432 (2004)

Mismatch Binding/Removal

bind MutS

removeMutS + mismatch

(error-free DNA)

Carr et al. NAR 32 (2004)

Mismatch Cleavage

Smith & ModrichPNAS 94 (1997)

1 error per 104

base pairs

error-corrected dna synthesis peter carr mit media laboratory

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