Product Overview
Custom cDNA Library Construction, Normalization
& Subtraction Services
• Our patented SMART™ cDNA
synthesis technology enriches
for full-length clones
You Provide Total or Poly A+ RNA
9Quality Checkpoint
• SMART technology maintains
original gene representation, even
when starting material is limited
During the SMART cDNA Synthesis, two Sfi restriction sites (A and B)
are incorporated via a modified Oligo d(T) primer and the SMART Oligo.
The entire cDNA pool is amplified by PCR using a primer that recognizes
both the 3' and 5' ends of every cDNA molecule. The result is a high-quality,
representative cDNA pool requiring only minimal amounts of RNA.
Conventional synthesis is also available.
• Normalization enhances the gene
discovery rate of cDNA libraries
and next-generation sequencing
• Sample integrity is checked
by gel profile
• rRNA content is determined
by capillary electrophoresis
9Quality Checkpoint
• Quality driven service—verification
of sample integrity performed
• Quality of cDNA is analyzed
by gel profile
SfiI Digestion and Ligation
Library Construction Service
Clontech offers custom, full-length cDNA
libraries, full-length normalized cDNA
libraries, and subtractive hybridization
services. All of these custom services undergo
rigorous quality verification at each milestone, resulting in quality you can rely on.
By ordering library construction or cDNA
subtraction services from Clontech, you
will not only simplify your research, but
you will also gain access to the scientists
who pioneered SMART cDNA Amplification and PCR-Select™ cDNA Subtraction.
These groundbreaking, patented Clontech
technologies are the basis of our custom
full-length cDNA library construction &
subtraction services.
• Concentration is checked
by UV spectroscopy
SMART™ cDNA Synthesis
SfiI digestion at the A and B sites creates sticky ends on the cDNA and prepares
it for directional ligation into the vector of your choice. Please see order
form for vector choices.
9Quality Checkpoint
Perform Packaging
Reaction
For λTriplEx2 libraries, DNA
and Lambda packaging reactions
are combined to create phage
particles containing clones
of library.
Transformation
For non-λTriplEx2 libraries, DNA
is transformed into E. coli and
spread on plates
• RsaI-digested and undigested
cDNA is visualized by gel
electrophoresis
• Subtracted and unsubtracted
cDNA is visualized by gel
electrophoresis
9Quality Checkpoint
• Insert screening analysis
is performed
Library Amplification
Screening results are analyzed and plasmid DNA is isolated from
differentially expressed clones.
• Recombination efficiency
is checked by performing
blue/white screen
9Quality Checkpoint
• Titer is determined
SMART Full-Length
cDNA Library
Construction Service
cDNA libraries constructed using our
proprietary SMART (Switching Mechanism At 5' end of the RNA Transcript)
technology contain a higher percentage
of full-length clones than do libraries
constructed either by conventional methods
or other full-length cDNA synthesis protocols.
SMART™ is a unique technology that allows
the efficient incorporation of synthetic adaptors on both the 5' and 3' ends of cDNA
during first strand synthesis, without
adaptor ligation. This enables end-to-end
PCR and prevents the generation of chimeric
cDNA inserts, resulting in a clean and
representative cDNA population. Truncated
products resulting from premature termination of the reverse transcription reaction,
as well as genomic DNA, generally do not
Figure 1. Custom cDNA Library Construction Service. This figure illustrates the steps taken
by Clontech’s experts to construct your custom SMART cDNA library. The service guarantees
delivery of at least 1 x 106 independent clones. Turn around time is dependent on the starting
material and can be anywhere from 8 –10 weeks. To order a custom library, contact your
local Territory Manager, and complete the Custom Library Service order form available
at www.clontech.com
incorporate the SMART adaptors, and
consequently are not amplified during PCR.
The highly efficient SMART cDNA
synthesis procedure allows us to amplify
cDNA from very little starting material,
and from a variety of sources. We accept
total or poly A+ RNA, tissue, or cell samples.
As little as 5 μg of poly A+ RNA, or 15 μg
of total RNA is sufficient for generating
highly representative cDNA to construct
your custom library (Figure 1). Libraries
can be constructed in the following vectors:
λTriplEx2, pGADT7-RecAB (Matchmaker™
Libraries), pRetro-Lib (Retroviral), pDNR-
2 2 Clontech Laboratories, Inc. • www.clontech.com
Lib (Creator™ System), and more. We
deliver libraries of at least 1 x 10⁶ clones
in 8–10 weeks.
Normalized FullLength cDNA Library
Construction Service
The wide variety of different mRNAs
represented in cells and tissues makes it
extremely difficult to analyze rare messages
in cDNA libraries. Therefore, for rare
transcript cloning and analysis, equilibration or normalization of highly abundant
and less abundant genes is required.
Clontechniques October 2008
Product Overview
Custom cDNA Library Construction, Normalization
& Subtraction Services…continued
A
M
1
2
B
kb
ACTB
M
1
5.0 –
4.0 –
3.0 –
kb
2.0 –
2.0 –
1.0 –
1.0 –
2
GAPD
M
1
2
0.75 –
0.5 –
Figure 2. Efficiency of DSN-Normalization. Panel A. DSN-Normalization equalizes transcript
abundance while maintaining average cDNA length. Agarose gel electrophoresis reveals
bands corresponding to abundant transcripts in non-normalized SMART-amplified cDNA
(Lane 1) are apparently removed in the DSN-normalized, SMART-amplified cDNA sample
(Lane 2); human placenta total RNA was used as starting material. Lane M: 1 kb DNA size
marker. Panel B. Specific reductions in the levels of two abundant transcripts are demonstrated by virtual Northern analysis of non-normalized (Lanes 1) and DSN-normalized
human placenta cDNA (Lanes 2). GenBank Accession numbers: GAPD, NM_002046; ACTB,
NM_001101. Lane M: DNA Size Marker.
Normalization significantly reduces the
number of clones obtained from the highly
abundant transcripts of housekeeping genes,
allowing you to screen for clones of medium
and low abundance transcripts in less time
and with less effort. We recommend the
use of normalized libraries, as they contain
a more diverse population of clones,
and reduce the screening bias for clones
derived from highly abundant transcripts,
significantly increasing the ability to
detect your clones of interest.
We start our Custom Normalized Library
Service by generating SMART amplified
full-length cDNA from your RNA sample.
We then normalize the cDNA population
using duplex-specific nuclease (DSN)
normalization, a proprietary technology
that is especially effective for the discovery
of rare genes (1, 2). DSN-normalization
equalizes the abundance of all transcripts
in a sample by specifically reducing the
proportion of those that are highly abundant,
consequently enhancing the gene discovery
rate of cDNA libraries and next-generation
sequencing. The efficiency of DSN-normalization is demonstrated by comparing
DSN-normalized and non-normalized
SMART-amplified, full-length cDNA
(Figure 2).
We check the efficiency of cDNA normalization by virtual Northern analysis (3), as
shown in Figure 2, Panel B. You can send
at least one abundant transcript sequence
and up to three probes (nonmammalian
species) corresponding to abundant
transcripts (1 μg each) for normalization
efficiency analysis. Only two of the probes
will be used. Probes prepared by PCR
amplification or by restriction enzyme
digestion of plasmid DNA are appropriate.
For human, mouse, or rat housekeeping
genes (i.e. β-actin, ubiquitin or G3PDH),
in-house probes will be used.
Last but not least, we clone the normalized
cDNA into your choice of vector. If you
would like uncloned, normalized cDNA
for further 454 sequencing or other nextgeneration sequencing, the option is available
on request. At the completion of our Custom
Normalized Library Service, we deliver normalized SMART cDNA; non-normalized
SMART cDNA; the normalized cDNA library
in E. coli DH10B or λ lysate (1 x 10⁶ independent clones); and the results of the virtual
Northern analysis. Additionally, we provide
all data generated during the process.
Clontech Laboratories, Inc. • www.clontech.com
PCR-Select cDNA
Subtraction Service
Clontech’s subtraction experts can transform
your RNA into purified and sequenced,
differentially expressed cDNAs in a matter
of weeks (Figure 3). PCR-Select Custom
cDNA Subtraction enriches for differentially expressed genes using the innovative
Suppression Subtractive Hybridization (SSH)
method, originally described by Diatchenko
et al. (4). Depending on your particular needs,
we offer two levels of service. Level I Service
includes construction of forward and reverse
subtracted libraries and the initial stages of
differential screening. Level II Service
includes everything in Level I, and completes
differential screening for the forward
subtracted library. For samples with very
similar expression profiles (which often
generate high levels of background in
subtractions), we offer Mirror Orientation
Service (MOS) for superior identification
of differentially expressed genes. We deliver
your pooled, amplified and subtracted library
in E. coli; forward and reverse subtracted
libraries arrayed on five 96-well plates; or
a single library arrayed on 10 plates; and
differentially expressed clones transformed
in purified plasmids. Additionally, we provide
all data generated during differential
expression, virtual Northern analyses,
and BLAST searches.
Order Clontech
Custom Services—
Your Way
We invite you to bring your custom service
needs to us, whether based on our existing
products or custom derivations of our
standard platforms tuned to your specific
needs. We provide a free consultation with
a member of our Custom Services Team
during which feasibility of your experiment,
sample requirements, turnaround times,
and the nature of the deliverables are
discussed. Allow our talented group of
scientists to help design the best, most
Clontechniques October 2008 2 3
Product Overview
Custom cDNA Library Construction, Normalization
& Subtraction Services…continued
Service
You Provide Total or Poly A+ RNA
9Quality Checkpoint
cDNA Synthesis
SMART cDNA is synthesized from customer-supplied RNA. In SMART
cDNA synthesis, oligos containing the RsaI restriction site are incorporated
at both the 3' and 5' ends of each cDNA molecule, with no adaptor ligation.
The entire cDNA pool is amplified by PCR using a primer that recognizes
both the 3' and 5' ends of every cDNA molecule. The result is a high-quality,
representative cDNA pool requiring only minimal amounts of RNA.
Clontech PCR-Select™ cDNA Subtraction Service
Subtraction
Custom Creator cDNA Library in pDNR-Lib
630019
• Sample integrity is checked
by gel profile
Custom SMART cDNA Library in λTriplEx2,
unamplified only
630020
• rRNA content is determined
by capillary electrophoresis
Custom SMART cDNA Library in λTriplEx2,
amplified only
630021
9Quality Checkpoint
Custom SMART cDNA Library in λTriplEx2,
amplified & unamplified
630022
• Quality of cDNA is analyzed
by gel profile
Custom Large-Insert cDNA Library in λTriplEx2,
amplified only
630024
Custom Pretransformed Matchmaker
cDNA Library Construction in pGADT7-Rec/Y187
9Quality Checkpoint
The subtracted cDNA pool is ligated into a vector and transformed
into E. coli to create the subtracted library, arrayed in 96-well plates.
• RsaI-digested and undigested
cDNA is visualized by gel
electrophoresis
• Subtracted and unsubtracted
cDNA is visualized by gel
electrophoresis
Differential Screening Analysis
Level I Service (Cat. No. 630025)
One plate from each forward
and reverse-subtracted library
is screened using forward
and reverse subtracted probes.
Level II Service (Cat. No. 630026)
Five plates from either forward
or reverse subtracted library
and one plate from the library
subtracted in the opposite direction
are screened using forward and
reverse subtracted probes.
Screening results are analyzed and plasmid DNA is isolated from differentially
expressed clones.
630036
Custom Pretransformed Matchmaker
cDNA Library Construction in pDNR-GADT7-Rec/Y187
630045
Custom Pretransformed Matchmaker
cDNA Library Construction in pDNR-GADT7-Rec/AH109
630047
Matchmaker cDNA Library in pGADT7-RecAB
630058
Library Custom (Generic)
630053
Normalized Custom cDNA Library Construction Service
630064
Custom PCR-Select Subtraction: Level I
630025
9Quality Checkpoint
Data Analysis and Clone Isolation
630017
• Concentration is checked
by UV spectroscopy
The cDNA is digested with RsaI to create short, similarly-sized cDNA
fragments which are then ligated with adaptors. Following subtractive
hybridization, up-regulated genes are enriched by PCR.
Library Construction
Cat. No.
Custom cDNA Library Construction in pRetro-Lib
• 5 clones are subjected
to virtual Northern blot
analysis to confirm
differential screening results
Custom PCR-Select Subtraction
& Differential Screening: Level II
630026
MOS for Custom PCR-Select
630044
Notice to Purchaser
Sequencing and BLAST Search
Differentially expressed clones are sequenced and subjected to NCBI’s
BLAST (Basic Local Alignment Search Tool) Search. Please see reverse
for deliverables.
Figure 3. Clontech Custom PCR-Select cDNA Subtraction Service. This figure illustrates
the steps taken by Clontech’s experts to perform the PCR-Select cDNA subtraction, library
construction, and differential screening for Level I and Level II Services. Turnaround time
for Level I Service is 14–16 weeks. Turnaround time for Level II Service is 17–19 weeks.
To order Level I or Level II Service, contact your local Territory Manager, and complete the
Clontech PCR-Select Subtraction Custom Service order form available at www.clontech.com
cost-effective solutions for your research.
For more information on Clontech’s
Custom Services, including overviews,
order forms, and related product information, please visit the Custom Services
section of our website.
Please see the PCR-Select™ cDNA Hybridization,
Creator™ and Creator™ Access Services,
Matchmaker™ Two-Hybrid System, Retroviral
Vectors, SfiI Cloning Strategy, and SMART™
Amplification Products licensing statements
on page 25.
References
1. Zhulidov, P. A. et al. (2004) Nucleic Acids Res.
32(3):e37.
2. Shagin, D. A. et al. (2002) Genome Res.
12(12):1953–1942.
3. Franz, O. et al. (1999) Nucleic Acids Res.
27(11):e3.
4. Diatchenko, L. et al. (1996) Proc. Natl. Acad.
Sci. USA 93(12):6025–6030.
2 4 Clontech Laboratories, Inc. • www.clontech.com
Clontechniques October 2008