Dynabeads® for specific capture (pdf) - Invitrogen

Tools for Nucleic Acid IVD
Powering reliability for IVD
assay development
Dynabeads ® for specific capture

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Tools for Nucleic Acid IVD
Specific capture solutions
Dynabeads ® magnetic separation technology
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Enhance the sensitivity of your assay
Streamline your workflows
Offer validated products for IVD
Are you thinking about developing or improving a test for specific
nucleic acid targets? Would you like to increase the specificity and
sensitivity, expand the quantification, and reduce cost per reac-
tion? Specific nucleic acid capture with Dynabeads® will provide
definite advantages and introduce efficiencies to your assay.
Reputation is earned
Dynabeads® are well established as the gold-standard method
for automated IVD sample preparation, and are integrated into
more than 25,000 IVD instruments worldwide.
Leading IVD companies choose Dynabeads® because of our
ability to deliver IVD-grade products and ensure scale-up. As a
company, Invitrogen’s Dynal® division can follow your projects
from the early phases of R&D, through assay development and
validation, into the IVD assay market.
The IVD industry faces specific regulatory requirements.
Invitrogen’s Dynal® division meets these today by complying with
ISO 9001:2000 and ISO 13485:2003, and follows the requirements
of cGMP when necessary. Furthermore, our quality system is
based on customer requirements, enabling us to offer custom QA
to support customers’ own GMP initiatives or local regulations.
The tightly controlled production processes yield uniform spheri-
cal beads (Figure 1) with highly defined product characteristics.
This ensures consistency in:
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Size and surface area
Surface chemistry
Nucleic acid binding capacity
Magnetic particles from alternative suppliers often have a
random size range distribution and surface area that could com-
promise the reproducibility of your assay results. 1
Figure 1—Dynabeads® are monosized. The defined characteristics and unique
level of conformity (both within and between batches) ensure consistency
and will contribute to highly reproducible assay results. 1

Dynabeads® make a difference
Magnetic separation is very robust and flexible (Figure 2), and
has revolutionized sample preparation in automated systems.
With manual intervention greatly reduced, skilled technicians
can drive more results per day when compared to other
techniques (Figure 3).
Choice of capture strategy
To meet the specific requirements of your sample preparation
and facilitate assay improvements, a range of Dynabeads® are
available:
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Dynabeads® Oligo(dT) 25 —for isolation of mRNA, solid phase
reverse transcription, amplification, and detection
Dynabeads® Streptavidin—for isolation of specific targets
via a biotinylated capture probe—to allow further flexibility,
four types of streptavidin-coupled beads are available
Dynabeads® with custom-coupled probe—for isola-
tion, solid-phase amplification, and detection of specific
sequences—optimized oligo probes are covalently coupled
to the beads on an OEM basis
Phenol extraction
20 min
Manual
loading
and lysis
5–10 min
Lysis and binding
5–10 min
Centrifugation
6 min
Centrifugation
20 min
Transfer
and wash
10 min
Chloroform process
20 min
Centrifugation
and wash
10–15 min
A. Indirect capture
TTACGTAA
AATGCATT
Biotinylated capture probe is
added to the sample prior to
addition of beads
TTACGTAA
AATGCATT
TTACGTAA
AATGCATT
AATGCATT
B. Direct capture
TTACGTAA
Specific capture probe
is covalently coupled
to the beads
TTACGTAA
AATGCATT
TTACGTAA
AATGCATT
Figure 2—Indirect and direct capture methods. Nucleic acids are isolated from
a lysed sample using either Dynabeads® Streptavidin and a biotinylated
specific capture probe (A) or a specific capture probe covalently coupled to
Dynabeads® (B).
Centrifugation
20 min
Traditional manual phenol/choloroform procedure (2–3 hr)
Spin column procedure (45–60 min)
Manual handling time up to 25 min
Magnetic
separation and
elution
15 min
Dynabeads ® (20–25 min)
Manual handling time

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Tools for Nucleic Acid IVD
Table 1—Dynabeads® selection guide.
mRNA
capture
Solid-phase cDNA
production
Viral DNA/RNA
capture from
blood
Solid-phase
amplification
Solid-phase
detection
Larger sample volume
(0.5–1.5 ml)
Dynabeads®
Oligo (dT) 25
Dynabeads®
Streptavidin
Customcoupled
Dynabeads®
Well-defined
primer set
PCR clean-up
Selection guide
The range of Dynabeads® products will allow you to stream-
line infectious disease testing or develop novel assays for gene
expression and cancer diagnostics (Table 1). Your specific selec-
tion strategy and assay design will determine which of the avail-
able Dynabeads® are most suitable. The table below will help you
select the optimal bead for your assay development or automa-
tion program.
Custom product development and optimization
What are the specific requirements for your assay? Do you need
a high level of specificity and sensitivity? What about time and
throughput? These specifications define the need for develop-
ment, automation, and streamlining of your workflows.
Specific primer or probe sequences, types of samples, and
target parameters often require optimization for a given assay or
instrument. Such optimizations can include buffer development,
larger sample volumes, or the parallel testing of different bead
types (to assess binding, motility, etc.). We can advise and support
the evaluation of Dynabeads® and ensure the required regulatory
compliance for your assays or automated instrument. Contact us
to find out how Dynabeads® can make a difference in your assays
or automated instruments.

Dynabeads® Oligo(dT) 25 for direct mRNA
capture and analysis:
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Captures a representative mRNA population
Offers the same mRNA yields as more labor-intensive methods
Provides fast, robust, and automatable protocols
The remarkable sensitivity allows even low copy number
transcripts to be reliably captured and detected, while providing
easy magnetic handling for streamlined workflows (Figure 4). With
no inhibition of enzymatic activity, mRNA isolation with Dyna-
beads® Oligo(dT) 25 is directly compatible with reverse transcrip-
tion and PCR.
Dynabeads® Streptavidin for indirect biotinmediated
capture and detection:
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High capacity and a wide dynamic range
Robust and flexible protocol development
Ideal for target sequence optimization
The indirect capture approach using a biotinylated probe is
often preferred when the target concentration is low, when the
specific affinity is weak, or when the binding kinetics are slow
(Figure 5). Dynabeads® Streptavidin are seen as the gold standard
and are widely used in the IVD industry.
C t
23.5
23.0
22.5
22.0
21.5
21.0
20.5
β-actin
medium size
high copy
XPO-1
larger size
low copy
POLR2
low size
medium copy
Figure 4—Representative mRNA population shown by RT-qPCR. Transcripts of different
lengths and abundances were isolated using Dynabeads® Oligo(dT) 25
(blue) and shown to be as effective as or better than (i.e., giving lower C t values)
two total RNA isolation techniques (gray: Invitrogen’s TRIzol® Reagent;
red: other commercially available product). The analyzed transcripts are
β-actin (1,793 bases long, 1,500 copies per cell in active tissue), exportin 1
(XPO-1, 4,148 bases, 129 copies) and RNA polymerase ll (POLR2, 392 bases,
800 copies). As confirmed by the RT-qPCR analyses shown, magnetic capture
with Dynabeads® gives a highly representative mRNA population for a range
of transcripts in terms of their size and abundance.
Oligonucleotide bound (pmol/mg)
600
500
400
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Oligonucleotide added (nmol/mg beads)
Figure 5—Highly consistent and predictable binding of biotinylated oligonucleotides.
The amount of biotinylated oligo capture probe bound to Dynabeads®
Streptavidin is controlled by varying the amount of probe added. An excess
of probe can be added to saturate the beads, but optimal assay performance
is typically achieved at relatively low concentrations of capture probe. The
amounts of Dynabeads® added were 5 mg/ml (red), 2.5 mg/ml (blue) and
1.7 mg/ml (gray).
www.invitrogen.com
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Tools for Nucleic Acid IVD
Custom-coupled Dynabeads® for direct coupling
of specific primer/probe:
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Process with reduced reagent usage, no biotinylation step
Use with well-defined probe/primer targets, e.g. conserved
regions of viral genomes
Allows capture under high organic salt conditions, e.g. GTC
Coupling of a specific capture probe will enhance solid phase
amplification and detection while streamlining workflows, and
offer economy of scale by removing the need for biotin reagents
and a separate supply of oligos (Figure 6).
1,000
800
600
400
200
Eluted ssDNA (RU) 1,200
0
0 200 400 600 800 1,000 1,200 1,400
Amount of beads (µg)
Figure 6—Linear binding and reliable specificity. A specific probe is coupled to
Dynabeads® and used for capture from a 100 µl sample containing 4 pmol of
a single-stranded (blue) and double-stranded (red) DNA fragment, 324 bp in
length. The predictable performance of probe-coupled Dynabeads® is documented
by the linear binding relationship between bead and target quantities.
The reduced yield of dsDNA compared to ssDNA is explained by the competition
between hybridization to immobilized capture probe and re-annealing of the
dsDNA sequence. A nonspecific probe serves as negative control in both setups.
The absence of significant binding confirms specificity (purple and green).
Advantages of magnetic separation
Dynabeads® magnetic separation technology is rapid, robust, and
reproducible. Larger sample volumes can be used to enhance
detection sensitivity, while the target can be concentrated and
aliquoted into one or more, smaller elution volumes for different
downstream applications. The technology is:
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Robust and easy to automate
Versatile, adaptable to multiplex formats
Ideally suited for high-throughput systems
Specificity, sensitivity, and reproducibility
The unique consistency of Dynabeads® ensures a high level of
reproducibility and drives reliability for your assays. Key advan-
tages are:
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Increased specificity as targets are selectively purified prior
to amplification
Increased sensitivity by eliminating human DNA in patho-
gen tests
A variety of clinical samples can be used without inhibiting
amplification
Dynabeads® make good business sense
In addition to thorough in-house product development, produc-
tion processes, and exacting quality control, the performance
and reliability of Dynabeads® has been extensively documented
by many users in numerous published articles. 2–9

Accelerate time-to-market for your assays
Dynabeads® are equally at home in a hands-on benchtop experi-
ment as they are in high-throughput automated systems. We
deliver the scale, product quality, and expertise to power your
assay from R&D to your finished diagnostic test.
Meeting the IVD challenge
Diagnostic companies strive to develop assays with improved
precision and sensitivity, while aiming for higher speed and
throughput. Reliability in terms of efficiency and reproducibility is
essential, as are the specific regulatory requirements.
Specific capture using Dynabeads® helps you overcome
these challenges. The kinetics, capacity, and reproducibility of
the beads contribute to the streamlining and automation of your
workflows. These are valuable advantages that will enhance the
development of new IVD processes and instruments. Invitrogen’s
Dynal division complies with ISO 9001:2000 and ISO 13485:2003
(Figure 7), and supplies IVD-grade products on an OEM basis.
Your innovation partner in IVD
Dynabeads® provide innovation, along with proven performance
and reproducibility for all your assays. As a preferred partner to
leading IVD companies, we develop robust IVD-grade products
from our existing biodiscovery portfolio, or by entering novel col-
laborations.
Contact us and let Dynabeads® magnetic separation tech-
nology help solve your assay development needs. If you would
like to discuss a potential collaboration or OEM agreement, please
contact us by email, dynal@invitrogen.com.
Figure 7—ISO certified and in line with US cGMP. Invitrogen’s Dynal division complies
with ISO 9001:2000 (since 1996) and ISO 13485:2003 (since 2003), and
follows the requirements of cGMP when necessary.
www.invitrogen.com
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References and further reading
1. In-house documentation (2002), Dynal Bead Based Separations, Invitrogen
Group.
Diagnostic research and assay development using Dynabeads® for detection
of viral pathogens:
2. Stelzl, E. et al. (2002) Evaluation of an automated sample preparation protocol for
quantitative detection of hepatitis C virus RNA. Journal of Clinical Microbiology
40(4):1447-1450.
3. Meng, Q. et al. (2001) Automated multiplex assay system for simultaneous
detection of hepatitis B virus DNA, hepatitis C virus RNA and human
immunodeficiency virus type 1 RNA. Journal of Clinical Microbiology 39(8):2937-
2945.
4. Böni, J. et al. (2004) Detection of low copy numbers of HIV-1 proviral DNA in
patient PBMCs by a high-input sequence capture PCR (Mega-PCR). Journal of
Medical Virology 72(1):1-9.
5. Stevens, S.J.C. et al. (1999) Monitoring of Epstein-Barr virus DNA load in peripheral
blood by quantitative competitive PCR. Journal of Clinical Microbiology
37(9):2852-2857.
Recent research, new assays, and combined cell-enrichment strategies:
6. Wang, B. et al. (2005) Rapid and sensitive detection of severe acute respiratory
syndrome coronavirus by rolling circle amplification. Journal of Clinical
Microbiology 43(5):2339–2344.
7. Cornelissen, M. et al (2003) Gene expression profile of AIDS-related Kaposi’s
sarcoma. BMC Cancer online 3:7.
8. Zieglschmid, V. et al. (2005) Combination of immunomagnetic enrichment
with multiplex RT-PCR analysis for the detection of disseminated tumour cells.
Anticancer Research 25(3A):1803-1810.
9. Reinholz, M.M. et al. (2005) Evaluation of a panel of tumour markers for
molecular detection of circulating cancer cells in women with suspected breast
cancer. Clinical Cancer Research 11(10):3722-3732.
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