In addition to a standard gene synthesis service, IDT offers a novel, rapid, and reliable method to build and clone the genes you need at a fraction of the cost of full gene synthesis services. gBlocks® Gene Fragments are double-stranded, sequence-verified DNA blocks of length 125–750 bp. Their high sequence fidelity and rapid delivery time make gBlocks Gene Fragments ideal for a large range of synthetic biology applications. In this presentation, Dr Adam Clore reviews a variety of uses of gBlocks fragments, including CRISPR-based genome modification, qPCR and HRM controls, and the assembly of gene fragments using the Gibson Assembly® Method.

1. Beyond Cloning: 101 Uses of Synthetic, High-Fidelity,
Double-Stranded DNA
Integrated DNA Technologies
Adam Clore, PhD

2. gBlocks® Gene Fragments Product Information
 Double-stranded, linear, synthetic
DNA fragments
 200 ng DNA provided, dry
 Typically shipped within 2–4 business days
 Affordable for basic research needs
 Designed and tested with the Gibson
Assembly® method
 Suitable for all purposes that require dsDNA

3. Making gBlocks® Gene Fragments
 Assembled using IDT Ultramer® Oligonucleotides
 Correctly assembled sequences are enriched using a proprietary,
cloning-independent method
 Each gBlocks Gene Fragment is verified
 The result is high-fidelity, double-stranded DNA that is routinely
cloned to yield >80% correct colonies
Gene Assembly
• Leverages IDT
proprietary
Ultramer®
synthesis
technology
Assembly
Selection
• Process
removes gene
assemblies
with deletions
and/or
substitutions
Sequence
Confirmation
• Each gBlocks
fragment is
confirmed by
three
independent
methods
Ship Preparation
• gBlocks
fragements
are amplified,
normalized,
packaged, and
shipped

4. 3 Ways We Make Sure Your gBlocks are Correct
 Capillary Electrophoresis
 Mass Spectrometry
 Sanger Sequencing

5. Cloning of gBlocks® Gene Fragments
% WT/Full Coverage
120%
100%
80%
60%
40%
20%
0%
125bp
300bp
500bp
Length of gBlocks Fragment
750bp

6. The Evolution of gBlocks® Gene Fragments
— Cheaper, Faster, Better
Feb 2014
Dec 2013
•gBlocks Libraries
Nov 2013
Oct 2013
•750 bp for $149
•3–4 day TAT
Jan 2012
•500 bp
•4–7 day TAT
•$99
• Mass Spec QC
added
•2–4 day TAT
•$89/$129
•1 kb gBlocks
fragments

7. gBlocks® Gene Fragments Libraries




Pools of gBlocks Gene Fragments with 1–18 N or K mixed bases
Mixed bases need to be consecutive
Mixed bases need to be 125 bp from either end
Length of gBlocks libraries is between 251 and 500 bp
At least 125 bp
1–18 bases
At least 125 bp
NNNNN….NNNNN
Or
NNKNN….NKNNK
Top 4 Questions:
- Can you make more complex libraries?
Other mixed bases than N or K
Multiple variable regions
Variable regions within the first and
last 125 bp of the gBlocks
Specific codon substitutions (AlaΔSer)
………
- Why only 18 Ns?
- Are your libraries biased?
- Can I get a discount?
Libraries @idtdna.com

8. How are gBlocks® Libraries Made and QCed?
 How are they made?
 This is proprietary, but the process is based on our capability for making very
high quality oligos and gBlocks Gene Fragments.
 How are they QCed?
 The constant regions are gBlocks Gene Fragments and are QCed by size
verification, capillary electrophoresis, and mass spec for sequence verification.
 We rely on a validated process (by NGS) to ensure that >80% of DNA species are
present in the final 200 ng of material shipped.

9. Sequence Fidelity in the Constant Regions
5 NNK
6 NNK
Looking for the error rates at each position in the constant region of the gene fragments

10. Base Distribution in Variable Region
115 bp
115 bp
1 NNK
2 NNK
3 NNK
4 NNK
5 NNK
6 NNK
Count of reads by position
Position
Base mix
A
C
G
T
1
N
2502744
2183361
2124224
2761775
2
3
N
K
2400917
2286
2088264
3160
2315143 4180965
2767780 5385693
Percentage of each base by position
Position
Base mix
A
C
G
T
1
N
26%
23%
22%
29%
2
N
25%
22%
24%
29%
3
K
0.02%
0.03%
44%
56%
• Built 6 gene fragments
with 1–6 NNK codons
• Sequence-verified each
by NGS: MySeq®, 250 bp
reads, forward and
reverse

11. Base Distribution in Variable Region
6 NNK

12. Examples of Orders/Applications
•
•
•
•
•
•
NNK(1-18)
NNM(1-18)
Binding site engineering
Catalytic site analysis
Antibody engineering
Vaccine development
DNA binding analysis
Promoter optimization
NNK
• Systematic codon replacement
NNK(1-9)
NNK(1-9)
NNM(1-9)
• Introducing multiple variable regions
NNM(1-9)
Libraries@idtdna.com

13. Pricing and Delivery Time
Mixed Base
>1 billion sequence variants for = $1,439
TAT: 10–15 Business Days
# of Ns
Diversity
NNKs
Diversity
Price USD
0
1
0
1 $ 89.00
1
4
4 $ 239.00
2
16
16 $ 239.00
3
64
1
32 $ 314.00
4
256
128 $ 389.00
5
1,024
512 $ 464.00
6
4,096
2
1,024 $ 539.00
7
16,384
4,096 $ 614.00
8
65,536
16,384 $ 689.00
9
262,144
3
32,768 $ 764.00
10
1,048,576
131,072 $ 839.00
11
4,194,304
524,288 $ 914.00
12
16,777,216
4
1,048,576 $ 989.00
13
67,108,864
4,194,304 $1,064.00
14
268,435,456
16,777,216 $1,139.00
15
1,073,741,824
5
33,554,432 $1,214.00
16
4,294,967,296
134,217,728 $1,289.00
17
17,179,869,184
536,870,912 $1,364.00
18
68,719,476,736
6
1,073,741,824 $1,439.00

14. Remember the Carlson Curve?
 Compares the cost of reading DNA to the cost of writing DNA
• You get up to 418 combinations in a
tube = about 68 billion gene
fragments
• For $1,439
• That is $0.000 000 021 per gene
fragment
• Or 0.000 000 0042 ¢/base (for a
500 bp library)
1.0E-09
IDT

15. Biosecurity

IDT is one of the five founding members of the International Gene
Synthesis Consortium (IGSC)

Screens the sequence of every gene/gBlocks Gene Fragment order

To ensure safety and regulatory conformance

IDT reserves the right to refuse any order that does not pass this analysis

For more information about the IGSC and the Harmonized Screening Protocol, please visit the website
at http://www.genesynthesisconsortium.org/Home.html.
In October of 2010, the United States government issued final Screening Framework Guidance for
Providers of Synthetic Double-Stranded DNA, describing how commercial providers of synthetic genes
should perform gene sequence and customer screening. IDT and the other IGSC member companies
supported the adoption of the Screening Framework Guidance, and IDT follows that Guidance in its
application of the Harmonized Screening Protocol. For more information, please see 75 FR 62820 (Oct.
13, 2010), or http://federalregister.gov/a/2010-25728.


16. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

17. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP detection controls
New Technologies

18. 3 Ways to Assemble Genes
Ultramer® Oligos
Product Description
gBlocks® Gene Fragments
Custom Gene Synthesis
Single-stranded custom
oligo
Double-stranded linear
fragment
Double-stranded product
delivered in a vector/BAC
200 pmol
200 ng
>4μg
45–120 bases
125–750 bp
25 – 2M bp
2–3 business days
2–4 business days
Variable
Mass Spec
Sanger Sequencing
Sanger Sequencing (or
NGS for long constructs)
50–80%
85–90%
100%
288 oligos
1 fragment
1 gene
Delivery Amount
Length
Turnaround Time
Quality Control
Estimated Purity
Minimum Order Size
Sequence Fidelity
$
Cost
Delivery time
$$$

19. Gene Construction Case Study #1:
An alternative to site-directed mutagenesis
Direct cloning & mutagenesis
gBlocks® Gene
Fragments
gBlocks® Gene Fragments used as an
alternative to site-directed
mutagenesis to introduce 18 mutations
spread over the 1039 nt exon 7 of the
gene JARID2 in order to verify that C-rich
consensus sites with a central invariant
CA dinucleotide are important for the in
splicing of large exons >1000 nt.

20. Gene Construction Case Study #2:
Immune Response After Flu Vaccination
DECODED 2.4 (October 2012):
Using gBlocks® Gene Fragments to Generate
Antibody Variable Regions
Francois Vigneault, PhD
Church Lab at Harvard University
now Abvitro, Inc
Each domain (VL, VH, CL, CH) is
≈ 100 aa or ≈ 400 nt
So each domain = 1 gBlocks fragment

21. Identifying Rare Antibodies
1.
2.
3.
4.
5.
6.
Patient vaccination
Identification and quantification of all
mRNAs by NGS (multiple data points)—
thousands of antibody sequences
Select the very few VL and VH domains
that are highly expressed
Build the potentially best antibodies by
combining a small selection of VL
domains and gBlocks Gene Fragments
coding for the VH domains
Selection of the strongest binding
antibodies by phage display and surface
plasmon resonance (Georgiou lab at
University of Texas, Austin)
Verify when the best antibodies are
produced using NGS data

22. Making Gene Synthesis More Affordable
Assume 1200 bp gene; what is the price differential for 8 genes
with one variable region? Assume $0.35/bp
Genes
gBlocks® Gene Fragments
1
3’
1
5’
2
3’
2
5’
3
3’
3
5’
4
3’
4
5’
5
3’
5
5’
6
3’
6
5’
7
3’
7
5’
8
3’
8
5’
8 Genes = $3,360
10 gBlocks fragments = ~$890

23. Assembling Multiple gBlocks® With the Gibson Assembly®
Method
•
•
•
Gibson Assembly™ Master Mix

24. Gibson Assembly® Method
How Isothermal Assembly of gBlocks® Gene
Fragments Works
Step 1: gBlocks Gene Fragments are designed with
30 bp overlaps on the 3’ strand for use in the
reaction with the following steps.
Step 2: A mesophilic exonuclease briefly cleaves
bases from the 5’ end of the double-stranded
DNA fragments, before being inactivated by the
50°C reaction temperature.
Step 3: The newly generated, complementary,
single-stranded 3’ ends anneal.
Step 4: A high fidelity DNA polymerase fills in any
single-stranded gaps.
Step 5: Finally, a thermophilic DNA ligase
covalently joins DNA segments.

25. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

26. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

27. CRISPR — Easy Genome Modification
 Clustered Regularly Interspaced Short Palindromic Repeat
 A prokaryotic defense mechanism that screens for and cleaves specific DNA
sequences
 Can be used to create targeted changes to the genomes of
bacteria, archaea, and eukaryotes

28. The 3 Stages of CRISPR Resistance
●
Stage 1: CRISPR Adaptation
–
●
Stage 2: CRISPR Expression
–
●
Foreign DNA is incorporated in the CRISPR
array.
CRISPR RNAs (crRNAs) are transcribed from
CRISPR locus.
Stage 3: CRISPR Interference
–
Foreign nucleic acid complementary to the
crRNA is neutralized.

29. Utilizing CRISPR for Genome Modification
 We need 3 components:
1. CRISPR Associated Gene 9 (CAS9)
2. RNA with CRISPR repeats (crRNA)
3. Trans-acting RNA (tracrRNA)
* 2 and 3 can be combined into a
single sequence called a single guide
RNA (sgRNA)
Zhang lab: http://www.genome-engineering.org

30. CRISPR-Cas9 System in Mammals

31. gBlocks® Gene Fragments for CRISPR

32. gBlocks® Gene Fragments for CRISPR

33. Genome Editing Case Study #1:
CRISPR Mediated Deletions
Non Homologus End Joining (NHEJ)
Error prone
Leads to indels and rearrangements

34. Gene Fragments Used in CRISPR Research
• gBlocks U6-gRNA
• gBlocks T7-gRNA for IVT
4 gBlocks for
Cas9 codon
optimization

35. Genome Engineering Case Study #2:
CAS9 as a Homing device
 Multiple, tuned, gene activation with nuclease-dead CAS9/gene promoter
fusion proteins
 Promoter gene and sgRNA were gBlocks fragments

36. 2013 Citations of CRISPR/Cas Genome Editing with gBlocks®
First author
Affiliation
Journal
Chen
UCSF
Cell
Malina
McGill
Genes Dev.
Mali
Harvard Med School (Church lab)
Science
Mali
Harvard Med School (Church lab)
Nature Biotech
Friedland
Harvard Med School (Church lab)
Nature Methods
Perez-Pinera
Duke
Nature Methods
Dickinson
Univ. North Carolina
Nature Methods
Gilbert
UCSF
Cell
Cheng
Whitehead/MIT
Cell Research
Waaijers
Univ. Utrecht
Genetics
Gratz
Univ. Wisconsin
Genetics
Bassett
Oxford
Biology Open

37. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

38. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

39. Synthetic Template Case Study #1:
gBlocks® Gene Fragments as DNA Standards
 Zymo Research
Decoded 3.3 (July 2013)
• gBlocks Gene Fragments as truly
un-methylated DNA standards
• PrimeTime® qPCR Assays for
multiplex analysis

40. Synthetic Template Case Study #2:
gBlocks® Gene Fragment as Synthetic Template in Multiplex PCR
A single DNA source for 4 different standard curves.
ACVR2B-LIMK1-ACVR1B-CDK7 wt
TCATACCTGCATGAGGATGTGCCCTGGTGCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCA
CAGGGACTTTAAAAGTAAGAATGTATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACT
TTGGCTTGGGAACATCATCCACCGAGACCTCAACTCCCACAACTGCCTGGTCCGCGAGAACA
AGAATGTGGTGGTGGCTGACTTCGGGCTGGCGCGTCTCATGGTGGACGAGAAGACTGTATGT
GATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGAT
GGGGAAGATGATGCGAGAGTGTTGGTATGGATGTATGGTGTAGGTGTGGACATGTGGGCTGT
TGGCTGTATATTAGCAGAGTTACTTCTAAGGGTTCCTTTTTTGCCAGGAGATTCAGACCTTG
ATCAGCTAACAgcggccgc
• Equimolar ratios of the four samples are always perfect

41. gBlocks® Gene Fragments as Quadruplex Standards
Cq Values
gBlocks Fragments as Standards
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Fourplex Reaction Conditions
Hs LIMK1
Hs CDK7
Hs ACVR1B
Hs ACVR2B
2.00E+06
2.00E+04
Copies
2.00E+02
Reagent
10X buffer
100 mM dNTPs
50 mM MgCl2
25 µM Forward Primer 1
25 µM Reverse Primer 1
12.5 µM Probe
25 µM Forward Primer 2
25 µM Reverse Primer 2
12.5 µM Probe
25 µM Forward Primer 3
25 µM Reverse Primer 3
12.5 µM Probe
25 µM Forward Primer 4
25 µM Reverse Primer 4
12.5 µM Probe
Immolase polymerase
H2O
Template
Final Conc.
1X
800 nM
3 mM
500 nM
500 nM
250 nM
500 nM
500 nM
250 nM
500 nM
500 nM
250 nM
500 nM
500 nM
250 nM
0.8 U
----

42. Synthetic Template Case Study #3:
Using gBlocks® Gene Fragments As Modified Standards
While both template sequences contain the primers and probe binding sites, by altering the
length of one, the modified amplicon can be distinguished from the endogenous one.
Hs.PT.51.4056836 LIMK1
Hs LIMK1 Forward
GAACATCATCCACCGAGACC
Hs LIMK1 Reverse
AGTCTTCTCGTCCACCATGA
HS LIMK1 Probe
CCAGCCCGAAGTCAGCCACC
Hs LIMK1 endogenous amplicon sequence
GAACATCATCCACCGAGACCTCAACTCCCACAACTGCCTGGTCCGCGAGAACAAGAATGTGGTGGTGG
CTGACTTCGGGCTGGCGCGTCTCATGGTGGACGAGAAGACT
Hs LIMK1 –10
GAACATCATCCACCGAGACCTCAACTCCCACAACTGCCTAACAAGAATGTGGTGGTGGCTGACTTCGGG
CTGGCGCGTCTCATGGTGGACGAGAAGACT

43. SYBR® Green Dye Dissociation Curve
gBlocks fragment (endogenous )
By deleting or adding bases, a unique
standard can be used that is
distinguishable from the endogenous
sequence.
gBlocks fragment (–10 bases)
If you have trouble with
contamination, you will always be able
to distinguish the standard from the
endogenous amplicon.

44. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

45. How are Researchers Using gBlocks® Gene Fragments?
Gene Construction and Modification
Genome Modification
qPCR and SNP Detection Controls
New Technologies

46. New Uses for gBlocks® Gene Fragments in 2014
 Gene variant libraries
 Promoter variation
 Gene insertion without homologous recombination

47. Synthetic Biology Partners
New England BioLabs
Gibson Assembly™ Master Mix
IDT and SGI are working
together to develop further
enabling tools for the SynBio
community.
Gene constructs from 5 kb to 2 Mb