Download Rapid Cloning of Antibody Variable Regions Using SMART

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Rapid Cloning of Antibody
Variable Regions Using
®
SMART Technology
SMARTer
RACE 5'/3' Kit
SMARTer® RACE cDNA Amplification
• Synthesize cDNA fragments containing full-length
sequences of variable regions
• Easily and accurately clone and analyze diverse
antibody genes
• Improve RACE PCR experiments with easier handling
and ligase-free cloning
Full-Length cDNA Synthesis Gets an Upgrade
Monoclonal antibodies have been widely used for various research and diagnostic
aims where strict antigen specificity is a key point of interest. Additionally, the
direct use of monoclonal antibodies as therapeutic treatments is becoming more
feasible due to a combination of advances in antibody engineering (e.g., the
development of chimeric and humanized antibodies) and improved technology for
antibody production.
Because antibody-antigen specificity is determined by the specific structure of
the antibody’s variable region, analysis of this region’s sequence is the first step
in many applications, including antibody production in an animal-free system,
development of antibodies as pharmaceuticals, and production of engineered
antibody variants. With SMART cDNA synthesis technology and a streamlined
method, SMARTer RACE kits provide a rapid, easy way to analyze these variable
and important nucleotide sequences.
Solutions for Sequence Analysis of Variable Domains
The immune system produces 108 varieties of antibodies to defend against
invading substances from the outside world. This diversity is achieved through
V(D)J recombination that occurs in developing lymphocytes. Antibody molecules
have heavy (H) and light (L) chain components with both constant and variable
(V) regions (Figure 1, next page); V(D)J recombination is a nearly random
rearrangement of the variable sequence with other gene segments. This process
generates unique antigen receptors. Since it is the variable region on the
N-terminal side of the H and L chains that determines antibody-antigen affinity,
analysis of this region is of great interest.
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are trademarks of TAKARA HOLDINGS, Kyoto, Japan. Clontech, the Clontech logo, In-Fusion, SeqAmp, SMART, SMARTer, SMARTScribe, Stellar, and
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incorporates Clontech’s unique SMART (Switching
Mechanism at the 5' end of RNA Template)
technology, which allows the reverse transcriptase
to reach the absolute 5' end of the transcript.
Most other RACE PCR methods do not capture
the 5' end, and therefore miss out on important
information. Furthermore, addition of the SMART
sequence at the 5' end enables the use of this site for
downstream amplification and cloning. Optimization
of the SMARTer RACE components and protocol
dramatically reduce non-specific background and
increase amplification efficiency. The robust system
yields reliable results even with samples that are
contaminated with genomic DNA, and is appropriate
for use in a wide range of applications. Amplification
of cDNA fragments with a high-fidelity polymerase
ensures the accurate replication of nucleotide
sequences needed for effective analysis.
Variable domain
(VH)
Variable
domain
(VL)
L chain
Variable
region
Constant
region
Fab region
C terminal
H chain
C O N S T R U C T I O N
Fc region
C terminal
Figure 1. Basic antibody structure.
When analyzing the variable region, designing
universal primers that will amplify all possible
antibody sequences is extremely difficult. The
inherent variability in the upstream 5' terminus
of the H and L chains results in low sequence
homology. An effective way to analyze these
variable domains is to design a degenerate primer
in a constant region downstream of the variable
region, where the sequence is relatively conserved.
A target region can then be cloned using the 5' RACE
(Rapid Amplification of cDNA Ends) method, and
subsequently sequenced. SMARTer RACE kits bring
complete 5'-end information and sensitivity to
this process.
Rapid Cloning of Antibody Genes Derived
From a Mouse Hybridoma
In preparation for cloning mouse hybridoma antibody
genes, a primer for reverse transcription (RT primer)
and a reverse primer for RACE PCR (5' RACE primer)
were designed for H, L (κ), or L (λ) chains based on
the sequence of all mouse IgG classes registered in
the NCBI nucleotide database. By using the SMARTer
RACE method to perform first-strand cDNA synthesis
from total RNA, directly followed by 5'-RACE PCR
(Figure 2), it is possible to obtain not only 5'-terminal
sequences, but also cDNA fragments that contain fulllength sequences of the variable regions.
Advantages of SMARTer Technology in
RACE Applications
With the SMARTer RACE cDNA Amplification Kit, the
SMARTer II A Oligonucleotide is added to the end of
the cDNA during reverse transcription. This process
total RNA
XXX
5'
AAAAA 3'
RT primer
For H, L (κ), or L (λ) chain
5'
RT reaction
SMARTer II A Oligo
5'
3'
XXX
XXX
5'
AAAAA 3'
Universal Primer long
3'
RACE reaction
XXX
5'
Universal Primer short
5'
3'
5'-RACE primer
For H, L (κ), or L (λ) chain
Cloning
Figure 2. Experimental workflow for 5'-RACE PCR of the antibody variable domain using the SMARTer RACE cDNA Amplification Kit.
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A mouse hybridoma strain was cultured and
confirmed to produce a monoclonal antibody with
H and L chains consisting of ɣ2a and κ chains,
respectively. Total RNA was prepared and used as
input for the SMARTer RACE cDNA Amplification Kit.
First-strand cDNA synthesis and 5'-RACE PCR were
performed according to the recommended protocols
for both the heavy (H) and light (L) chains, and
amplified products were confirmed by agarose gel
electrophoresis of a portion of the reaction mixture
(Figure 3).
H chain: ɣ2a
M
H
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A
B
H chain: ɣ1 L chain: κ
M
H
L
M
H chain: ɣ1 L chain: κ
M
H
L
M
L chain: κ
L
M
C
D
H chain: ɣ2b L chain: κ
M
H
L
M
H chain: ɣ1 L chain: κ
M
H
L
M
Figure 3. Confirmation of successful amplification of antibody
genes from mouse hybridomas.
Amplified products were cloned into pUC118 and
then sequenced. Sequences were confirmed to
contain ORFs corresponding to the amino acid
sequences of the H and L chains obtained from
analysis of the N-terminal amino acid sequence of the
monoclonal antibody.
We then performed the same type of gene sequence
analysis of the variable antibody region for six
additional hybridoma strains. PCR products were
obtained from all antibody subclasses (Figure 4).
Sequence analysis showed that 90% of the H-chain
clones and 50% of the L-chain clones accurately coded
for the N-terminal amino acid sequence for their
respective target antibody proteins.
In a few cases, clones had sequence homology to
the target antibodies, yet coded for ORFs unlikely
to encode functional antibodies due to the presence
of stop codons in their sequences. In addition, we
suspect one particular clone contained a transcript
from myeloma cells used for cell fusion, because the
sequence was obtained from multiple hybridomas.
E
F
H chain: ɣ2b L chain: κ
M
H
L
M
H chain: ɣ1 L chain: κ
M
H
L
M
Figure 4. Amplification of various subclasses of mouse
antibody genes.
Some hybridomas generated more clones with this
common sequence than sequence coding for a target
monoclonal antibody. Based on these results, it is
clear that the N-terminal amino acid sequence of a
target antibody should be analyzed and confirmed in
parallel with DNA sequence analysis.
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Further Improvements to SMARTer RACE
cDNA Synthesis, Including Streamlined
Cloning
In an effort to enhance the RACE procedure, we
recently made significant updates to our SMARTer
RACE kits. The data shown above was obtained
with the SMARTer RACE cDNA Amplification Kit, in
conjunction with PrimeSTAR® HS DNA Polymerase
for PCR, and the Mighty Cloning Reagent Set (Blunt
End) for ligation cloning. Since then, we have
developed the SMARTer RACE 5'/3' Kit (Cat. # 634858,
634859), a complete system with the same unique
SMARTer core technology, plus further refinements in
reaction conditions and for downstream cloning.
The new kit contains all the components (save genespecific primers) necessary to perform SMARTer
RACE experiments. It provides high sensitivity and
specificity, as well as low background, by pairing the
SMARTer II A Oligonucleotide with SMARTScribe™
Reverse Transcriptase. Robust PCR performance is
provided by SeqAmp™ DNA Polymerase, which is
especially useful when amplifying challenging targets
that are long or GC-rich.
Key among the components of the SMARTer
RACE 5'/3' Kit is the In-Fusion® HD Cloning Kit , which
allows for easy, accurate cloning of your 5'-RACE
fragments without the use of ligase. A linearized
vector compatible with In-Fusion technology is
provided in the kit, and the included primers are
designed specifically to match this vector. The InFusion Cloning method is highly efficient and wellsuited to all cloning applications. By pairing this
technology with the well-established SMARTer RACE
method, it only takes two days to go from RNA to
clones ready for analysis.
C O N S T R U C T I O N
improved affinity, or with dual specificity. Evaluation
of antibody variable regions is the first step toward
these and many other antibody modifications, and
SMARTer 5'-RACE PCR is an efficient method for
cloning these targets for sequencing purposes.
Methods
Each of the seven mouse hybridoma strains (all stored
at Takara Bio, Inc.) were cultured and confirmed via
subclass testing to produce monoclonal antibodies
with H and L chains consisting of ɣ2a and κ or λ
chains, respectively. Total RNA was prepared from
106 cells (one 10-cm dish) using a FastPure® RNA Kit,
yielding ~70 µg.
A reverse transcription (RT) primer and a reverse
primer for RACE PCR (5' RACE primer) were designed
for H, L (κ), or L (λ) chains based on the sequence
of all mouse IgG classes registered in the NCBI
nucleotide database. Following the protocol for the
SMARTer RACE cDNA Amplification Kit, first-strand
cDNA was synthesized using 500 ng of total RNA as a
template. For each sample, two reverse transcription
reactions were performed at the same time, using RT
primers for H and L chains, respectively.
1 µl Total RNA (500 ng/µl)
1 µl RT primer (12 pmol/µl)
1.75 µl Deionized H2O
3.75 µl Total
These reaction mixtures were incubated at 72°C for 3
min, followed by 42°C for 2 min in order to denature
the RNA. 1 µl of the SMARTer II A Oligonucleotide
(12 µM) was then added and mixed, for a total volume
of 4.75 µl. A Master Mix (as shown below) was
prepared for each cDNA synthesis reaction.
Summary
2 µl 5X First-Strand Buffer
SMARTer RACE cDNA synthesis provides a sensitive,
streamlined solution for full-length sequence analysis
of antibody variable domains. The diversity in these
variable regions, while critical for effective immune
response, presents a challenge to researchers looking
to analyze these gene segments for a wide range
of studies, including pharmaceutical development.
Having this sequence makes it possible to engineer
antibodies with improved in vivo stability, with
1 µl DTT (20 mM)
1 µl dNTP Mix (10 mM)
0.25 µl RNase Inhibitor (40 U/µl
1 µl SMARTScribe Reverse Transcriptase
(100 U/µl) (Cat. # 639536, 639537,
639538)
5.25 µl Total
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5.25 µl of Master Mix was added to each reaction
for a total of 10 µl. First-strand cDNA synthesis was
performed at 42°C for 90 min, followed by 72°C for
10 min. 50 µl Tricine-EDTA Buffer 1 was added to each
reaction, and the samples were stored at –20°C until
the RACE reactions were performed.
The amplified products from the 5'-RACE PCR were
confirmed by agarose gel electrophoresis (using 2%
NuSieve 3:1 Agarose) on a portion of the reaction
mixtures. Products were cloned into pUC118 using the
Mighty Cloning Reagent Set (Blunt End) (Cat. # 6027),
and analyzed via Sanger sequencing.
5'-RACE PCR was performed with PrimeSTAR HS DNA
Polymerase (Cat. # R010A, R010B) with the first-strand
cDNA generated above as template. Components
were added as follows:
The above experiments were performed with the
SMARTer RACE cDNA Amplification Kit in conjunction
with several additional products. The current SMARTer
RACE 5'/3' Kit contains all the necessary components
for RACE PCR:
10 µl 5X PrimeSTAR Buffer (Mg2+ )
4 µl dNTP Mixture (2.5 mM)
2.5 µl First-strand cDNA
5 µl 10X UPM (Universal Primer A Mix)
SMARTer RACE 5'/3' Kit Components
First-strand cDNA
synthesis reagents
SMARTer first-strand synthesis reagents,
5'- and 3'-RACE PCR universal primer mix,
control RNA & primers (primers have been
modified for use with this specific kit)
DNA polymerase
SeqAmp DNA Polymerase
In-Fusion Cloning
reagents
Linearized pRACE vector, In-Fusion HD
Cloning Kit (In-Fusion HD Enzyme Premix,
linearized pUC19 Control Vector,
Control Insert)
Competent cells
Stellar™ Competent Cells
Gel extraction kit
for PCR
NucleoSpin Gel and PCR Clean-Up kit
1 µl 5'-RACE primer (10 pmol/μl)
0.5 µl PrimeSTAR HS DNA Polymerase
(2.5 U/µl)
27 µl H2O
50 µl Total
The thermal cycler program was set as follows:
94°C
10 sec
30 cycles:
98°C 10 sec
60°C 5 sec
72°C 1 min
72°C
3 min
P R O D U C T S
Cat. #
Product
Package Size
634858
SMARTer RACE 5'/3' Kit
10 Rxns
634859
SMARTer RACE 5'/3' Kit
20 Rxns
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