Nucleic Acid Isolation.indd - Invitrogen

6. Nucleic Acid Isolation 

Isolation of Pure, Intact mRNA 71 

• Choosing the Right Product 71 

• Isolate mRNA From Any Sample 73 

• A Wide Variety of mRNA Applications 74 

Genomic DNA Isolation 78 


• A Variety of Sample Types and Downstream Applications 79 

Dynabeads ® Streptavidin for Superior Results 81 


• Selected Dynabeads ® Streptavidin Applications 82 

Dye Terminator Removal 85 

www.dynalbiotech.com 

Visit www.dynalbiotech.com for the latest product up-dates and references. 

69

Application Overview 

DNA Isolation 

mRNA Isolation 

Depletion 

Expansion 

Positive Isolation 

Negative Isolation 

Capture of Biotinylated Target 

70 

Organelle Isolation 

Specific Protein Isolation 

Samples: 

Whole Blood 

Cord Blood 

Mononuclear Cells 

Bone Marrow 

Buffy Coat 

Spleen 

Lymph Node 

Tissue Digest 

www.dynalbiotech.com

Nucleic Acid Isolation 

Isolation of Pure, Intact mRNA 

High quality, full length mRNA is necessary for meaningful results in any gene 

expression study. Therefore choosing the optimal mRNA sample preparation 

method is essential. Magnetic separation with Dynabeads ® Oligo(dT) 25 is a 

simple and rapid method to obtain pure, intact mRNA without any DNA or 

rRNA contamination (fig. 1). 

Main Advantages of Dynabeads ® mRNA Isolation: 

• Easy-to-use protocol. 

• mRNA isolation in only 15 minutes. 

• No centrifugation or precipitation. 

• Easily adaptable to most robotic systems. 

• Isolated mRNA representative and comparable to total RNA. 

The scalable protocols consist of only a few steps of pipetting and magnetic 

separation, with all operations performed in a single tube. This significantly 

reduces the risk of RNase contamination and loss of precious mRNA. The 

mRNA isolation protocols are scalable to meet your specific needs. Because 

the oligo-dT residues are covalently linked to the surface of the beads, the 

Dynabeads ® Oligo(dT) 25 can, if desired, be regenerated and reused at least 

four times. 

The mRNA isolation protocols do not require any centrifugation or 

precipitation steps. The method is therefore perfect for automation, and can 

be implemented on most liquid handling robotic platforms (see page 76). 

Choosing the Right Product 

Dynal Biotech supplies a variety of products and kits for isolation of mRNA, all 

with Dynabeads ® Oligo(dT) 25 as the key component (fig. 2). Up to 10 µg mRNA 

can be isolated per ml of Dynabeads ® Oligo(dT) 25 , assuming regeneration 

of the beads. The final yield will depend on tissue and cell type and the 

expression level of the mRNA. 

Table 1. Which product to choose for your mRNA isolations. 


71 

Animal / Plant tissue 

mRNA Isolation 

Cultured cells Total RNA 

Regenerate 

and reuse 

· 

· 

· 

· 

· 

· 

· c 

Pure cell population 

isolated with Dynabeads ® 

TTTTTTTTTTTT 

+ 

AAAAAAAAAAAA mRNA 

Northern blotting 

Dot/blot hybridisation 

Primer extension 

In vitro translation 

S1 nuclease assay 

Hybridisation probes 

DNA array/chip 

TTTTTTTTTTTT 

TTTTTTTTTTTT 

TTTTTTTTTTTT 

Elute 

(optional) 

Serum / Plasma 

Crude lysate 

containing 

poly A RNA 

TTTTTTTTTTTT 


TTTTTTTTTTTT 


Reverse 

transcription 

TTTTTTTTTTTT ................... 1st strand cDNA 


· 

· 

· 

· 

· 

· 

· 

.............. 

RT-PCR 

cDNA synthesis 

Solid-phase cDNA library 

SAGE-experiments 

RACE-experiments 

Subtractive hybridisation 

Differential display 

Fig. 1: Isolation of mRNA from different starting 

samples and for a wide variety of downstream 

applications. 

Product Prod. No. Starting sample Sample size 

Dynabeads ® Oligo(dT) 25 

610.02 

610.05 

610.50 

Any cells, animal- or plant tissue Scalable protocols 

Dynabeads ® mRNA Purification Kit 610.06 Total RNA 75 µg total RNA (scalable) 

Dynabeads ® mRNA DIRECT Kit 

610.11 

610.12 

Any cells, animal- or plant tissue 

Dynabeads ® mRNA DIRECT Micro Kit 610.21 Any cells, animal- or plant tissue 

≤ 2 x 10 7 cells 

≤ 200 mg animal tissue 

≤ 400 mg plant tissue 

1-2.5 x 10 4 cells 

≤ 5 mg animal tissue 

≤ 10 mg plant tissue 

+ 

6 

7 

Nucleic Acid Isolation

Isolate mRNA from Any Sample 

Mammals 

Species Starting Sample - Specific Tissue or Cell Type 

Bovine Corpus luteum 

Dog Leucocytes 

Gerbil Cochlear cells 

Guinea pig Organ of corti (inner ear), Spiral ganglion (inner ear) 

Human 

Mouse 

Pig Adipose tissue, Heart 

Rat 

Birds Pigeon Breast muscle, Frozen tissues, Liver 

Fish 

Amniocytes, B cells, Blastocysts, Blood, Bone marrow, Brain, Breast, Bronchoalveolar 

cells, Cartilage, CD34 + cells, Cervical cancer cells, Chondrocytes, Colon carcinoma 

cells, Cultured cells, Embryo, Fibroblasts, Glomeruli (kidney), Gut, Hair roots, Heart, 

Hepatocytes, Keratinocytes, Kidney, Langerhans cells, Leucocytes, Leukemia cells, 

Liver, Lung, Lymphoblasts, Lymphocytes, Lymphoid cells, Monocytes, Muscles, 

Neutrophils, Oocytes, Pancreas, Peripheral blood mononuclear cells (PBMC), Peritoneal 

exudate cells, Placenta, Platelets, Retina, Skin, Stem cells, T cells, Testicles, Tumours, 

Umbilical vein endothelial cells 

Brain, Cortex, Embryonic tissue, Epidermis, Eye, Fibroblasts, Germ cells, Glomeruli 

(kidney), Heart, Interscapular brown adipose tissue pad, Kidney, Kidney tubules, Liver, 

Lung, Retina, Skeletal muscle, Testicles 

Adrenals, Brain, Cerebral cortex (pre-optic area), Heart, Hypothalamus (brain), Kidney, 

Liver, Lung, Muscle, Neuronal cells (brain), Organ of corti (inner ear), Pancreas, 

Pituitary, Renal cortex (kidney), Spiral ganglion (inner ear), Spleen 

Trout Brain, Eggs, Fibroblasts, Liver, Muscle, Ovaries, Pronephros 

Zebrafish Embryo 

Amphibians Xenopus laevis Liver, Ovaries 

Molluscs Sea Urchin Blastocysts, Coelomocytes, Eggs 

Insects Drosophila melanogaster Ovary, Whole insects 

Plants 

Yeasts 

Parasites 

Viruses 

Arabidopsis thaliana 

Hordeum vulgare 

Brassica oleracea Leaves, Root, Stigma 

Lupinus angustifolius Cotyledon 

Flowers, Leaves, Roots, Rosette leaves, Seed, Silique, Stem, Stem leaves, 

Whole plants 

Embryo, Endosperm , Leaves, Root, Seed aleurone, Seed embryos, Seed endosperm, 

Seed 

Zea mays Embryos, Endosperm, Flowers, Leaves, Ovules, Root, Seed 

Nicotiana tabacum Bud, Leaf (nuclei), Leaves, Root (nuclei), Seed, Stem 

Pisum sativum Root apical meristems (nuclei) 

Solanum tuberosum Leaves, Stolon tips 

Picea ssp. Root 

Lycopersicon ssp. 

Candida tropicalis Cell culture 

Hansenula polymorpha Soil samples 

Saccharomyces cerevisiae Cell culture, Soil samples 

Cryptosporidium parvum Oocyst 

Schistosoma mansoni Whole worm 

HCMV Infected cells 

HIV-1 

HIV-2 Plasma 

HTLV-I/II Infected cells 

Human coronavirus Infected cells 

Poliovirus Infected cells 

Epidermal leaf cell (single cells), Flower bud, Flowers, Guard cell in leaf (single cells), 

Leaves 

Cells in broncho-alveolar washes, Cerebrospinal fluid, PBMC, Plasma, Serum, 

T lymphocytes cell line, CD4+ cells 

Table 2. An overview of some of the many sample types from which mRNA has been isolated using Dynabeads ® Oligo (dT) 25 . All information from published 

literature. For a full reference-list, please contact Dynal Biotech Technical Support. 

72 


Direct mRNA isolation with the Dynabeads ® mRNA DIRECT Kit* (Product 

no. 610.11/12) takes only 15 minutes from start to finish. The starting 

sample can be crude extracts of animal tissues, plants and cells. This new 

and improved kit contains 5 times more Dynabeads ® Oligo(dT) 25 . 

The protocol and kit format of the Dynabeads ® mRNA DIRECT Micro Kit 

(Product no. 610.21) is adjusted to small sample requirements, providing 

a fast and reliable way to combine direct mRNA isolation followed by direct 

RT-PCR amplification. 

The mRNA can alternatively be purified from total RNA with the Dynabeads ® 

mRNA Purification Kit (Product no. 610.06). A typical mammalian cell 

contains about 10 pg of total RNA, of which 1-5 % is mRNA. 

Dynabeads ® Oligo(dT) 25 (Product No. 610.02/05/50) is the main component 

in all of the above mentioned kits and is also available as a stand-alone 

product. The ease and speed of magnetic isolation and handling of mRNA 

provide a superior alternative to traditional methods. 

Isolate mRNA from Any Sample! 

As documented in a large number of scientific publications, Dynabeads ® 

Oligo(dT) 25 have been used to isolate mRNA from virtually any cell or tissue 

sample. Effective cell lysis and rapid handling make it possible to isolate 

mRNA from tough samples such as yeast cells, plant material with high 

starch and/or RNase content, paraffin-embedded tissues and even directly 

from human whole blood. 

The table on the facing page (table 2), lists some of the many different sample 

types where Dynabeads ® Oligo(dT) 25 have been used to isolate mRNA directly 

from a crude starting sample (all information from published literature). 


73 

Choose the Right Product 

New & 

improved 

Fig. 2: The true uniform size and shape of 

Dynabeads ® Oligo (dT) 25 provide rapid and 

efficient binding kinetics and highly reproducible 

results. 

6 

7 


A Wide Variety of mRNA Applications 

Fig. 3: Positive isolation of a specific cell type 

using antibody-coated Dynabeads ® , followed by 

mRNA isolation using Dynabeads ® Oligo(dT) 25 . 

MW 

MW 

. 

. 

+ 

+ 

Patient 1 Patient 2 Patient 3 Patient 4 

Fig. 4: Isolation of DNA and mRNA from the same 

blood samples using Dynabeads ® on the TECAN 

Genesis ® platform. A: CD19 PCR on genomic DNA. 

B: CD19 RT-PCR on mRNA. Both the DNA and 

mRNA isolations were performed on 3 parallels 

of 4 patient samples. 

CK19 

EPCAM 

Pure cells isolated with cell-specific Dynabeads Ò 

TTTTTTTT 

AAAAAAAAA 

Ò 

Isolation of mRNA with Dynabeads Oligo(dT) 25 

for use in further downstream manipulations 

Patient 1 Patient 2 Patient 3 Patient 4 

Number of cells: 1 1 5 5 10 10 

Fig. 5: Single cell RT-PCR sensitivity of mRNA 

isolated from 1, 5 and 10 SW480 carcinoma cells. 

Single cells were picked with a micromanipulator 

and mRNA isolated with Dynabeads ® Oligo(dT) 25 . 

The mRNA was then reverse transcribed while still 

attached to the beads and the resulting solidphase 

cDNA library included directly in a full PCR. 

The solid-phase cDNA library used for detection of 

the carcinoma cell specific CK19 transcript (upper 

panel) was reused as template also in a second 

round of PCR, this time detecting the carcinoma 

specific transcript EPCAM (lower panel). 

A 

B 


mRNA from Pure Cell Populations 

Molecular analyses are increasingly employed at the resolution of pure cell 

populations. Data from such homogeneous cell samples are much more 

informative than that from larger samples of mixed cells. 

As shown in the diagram on page 70, Dynabeads ® -based technology allows 

the combined isolation of pure cells, nucleic acids and proteins from the 

same sample. 

Dynal Biotech offers a wide range of Dynabeads ® pre-coated with specific 

antibodies against the major types of blood cells (chapter 3). In addition to the 

pre-coated Dynabeads ® , secondary-coated or surface activated Dynabeads ® 

can easily be coupled with your choice of antibody to isolate any specific cell 

type. This offers the unique possibility of combining separation of specific 

cell populations with mRNA isolation for downstream analysis (fig. 3). 

The scalable magnetic mRNA isolation technology simplifies automation of 

sample preparation protocols (fig. 4). 

mRNA from Single Cells 

It is often necessary or desirable to analyse gene expression with sensitivity at 

the single cell level, particularly if the amount of available material is sparse. 

Specific examples include embryology, neurology, stem cell studies, studies 

of small solid tumours or circulating micrometastatic cells, or cells obtained 

from fine needle aspirates or laser capture microdissection. 

Dynabeads ® -based technology enables molecular characterisation even down 

to single-cell profiling (fig. 5). Further examples of single cell profiling include 

the work by Karrer et al. Starting from single tomato epidermal cells, they 

isolated mRNA for construction of cDNA libraries (1). Klein et al. combined 

transcriptome and genome analysis of single micrometastatic cells using 

Dynabeads ® Oligo(dT) 25 (2). 

As described in chapter 3, pure cells for gene expression analysis can be 

obtained using cell-specific Dynabeads ® . Dynabeads ® Epithelial Enrich have 

been used to capture a single epithelial cancer cell spiked into 1 ml of blood. 

The mRNA was isolated from the positively isolated cell using Dynabeads ® 

Oligo(dT) 25 , reverse transcribed into a solid-phase cDNA library and the 

carcinoma cell specific transcripts CK19 and EPCAM detected by solid-phase 

RT-PCR. Konishi et al. have used Dynabeads ® to isolate single live human 

neurons (3). 

74 


mRNA for cDNA Libraries 

Dynabeads ® Oligo(dT) 25 are particularly useful for the construction of cDNA 

libraries (fig. 6), which can be used for cDNA amplification, cDNA cloning, 

RACE experiments and subtractive hybridisation. 

When preparing a solid-phase cDNA library, the mRNA is first captured 

onto the Dynabeads ® Oligo(dT) 25 . The beads are then added directly to the 

RT-reaction, where the bead-bound oligo-dT residues act as primers for 

first strand cDNA synthesis. As the operations take place on a solid-phase, 

changing of buffers is fast, simple and effective. 

The patented Dynabeads ® solid-phase cDNA synthesis technology is 

compatible with various commercially available cDNA synthesis kits. The 

beads do not inhibit enzymatic reactions, and bead-bound mRNA can be 

added directly to the RT-reaction without any diluting elution steps. Without 

eluting the mRNA from the beads prior to RT-PCR amplification, large numbers 

of cell clones can be rapidly screened for their expression of any gene. This 

makes it possible to generate solid-phase cDNA libraries even from a single 

cell (1). In addition, because the first strand cDNA is covalently linked to 

the beads, the cDNA library can be regenerated and reused for an almost 

unlimited number of times. 

mRNA for RT-PCR and Real-Time PCR 

Dynabeads ® Oligo(dT) 25 and the Dynabeads ® mRNA DIRECT Micro Kit 

offer a fast, simple and reliable way to combine mRNA isolation and RT-PCR 

or real-time PCR. The isolated mRNA is DNA-free as working with very small 

numbers of cells limits DNA contamination. 

Dynabeads ® Oligo(dT) 25 can be applied directly to PCR (or any other enzymatic 

reactions) as the presence of the beads does not inhibit or reduce the 

enzymatic activity. This is because the iron is sealed inside the Dynabeads ® 

by an inert polymer shell that prevents leakage and inhibition of enzymes. 

The benefit of adding the beads to the RT-PCR is that elution, and thereby 

dilution, of the mRNA is avoided. Dynabeads ® Oligo(dT) 25 also have negligible 

autofluorescence, and can therefore be applied directly in real-time PCR 

instruments such as the ABI Prism ® 7000 without interfering with the results 

of the analysis. 

mRNA for Northern Blotting 

Only 1-5% of the total cytoplasmic RNA in a typical eukaryotic cell is mature 

mRNA, and many transcripts occur in medium to low abundance. Using 

mRNA rather than total RNA for your Northern analysis will result in cleaner 

and more sensitive results. The highly purified and intact mRNA isolated 

with Dynabeads ® Oligo(dT) 25 is ideal for Northern blotting, and gives reduced 

background and increased intensity of signals (fig. 7). 

The mRNA can either be isolated directly from the cells or tissue with 

Dynabeads ® Oligo(dT) 25 or one of the Dynabeads ® mRNA DIRECT kits, 

or from total RNA with the Dynbeads ® mRNA Purification Kit. The kits are 

flexible and scalable to fit any sample size, and are therefore particularly 

useful for preparation of mRNA for Northern blotting from small amounts of 

sample material. 


75 


TTTTTTTTTTTT 

TTTTTTTTTTTT 

TTTTTTTTTTTT 

Isolate mRNA by 

magnetic separation 

TTTTTTTTTTTT 


Synthesize first-strand 

cDNA without eluting the 

mRNA off the Dynabeads ® 

TTTTTTTTTTTT ................. 1st strand cDNA 


Fig. 6: mRNA isolated from a starting sample is 

easily converted to cDNA, even without elution 

from the Dynabeads ® Oligo(dT) 25 . This enables 

further downstream manipulations of the resulting 

solid-phase cDNA library. 

Fig. 7: Unambiguous Northern analysis of timecourse 

expression of Cyclin D 1 mRNA in human 

airway smooth muscle with (T) and without (C) 

stimulation with thrombin for 2,4,8, or 16 hours. 

In the left panel, 5 mg total RNA was loaded per 

lane. In the right panel, mRNA (extracted from 

75 µg total RNA using the Dynabeads ® mRNA 

Purification Kit) was loaded per lane. Courtesy of 

E. Guida and A. Stewart, St. Vincents Hospital, 

VIC, Australia. 

6 

7 



A B 

Fig. 8: Functional genomics approach using 

Dynabeads ® to find target genes in testicular 

cancer. A: Microarray using input mRNA, shows 

either up- (red) or down- (green) regulated genes 

in tumour relative to normal testis. B: Two of the 

genes were also validated as up-regulated at 

their protein level. Courtesy of R. Skotheim and 

R. A. Lothe, the Norwegian Radium Hospital, 

Oslo, Norway. 

Fig. 9: The Dynabeads ® mRNA isolation method 

is easily adapted to commercially available 

liquid handling platforms. Protocols have been 

developed for the Biomek ® 2000 from Beckman 

Coulter and the Tecan Genesis ® from Tecan AG. 

mRNA for Microarrays 

Dynabeads ® Oligo(dT) 25 are perfect for purifying mRNA for expression profiling 

applications. Direct mRNA isolation eliminates the background noise often 

observed when preparing cDNA probes from purified total RNA. 

The hybridisation kinetics between mRNA and the Dynabeads ® are extremely 

fast and efficient, and comparable to that of free solution hybridisation. This 

ensures negligible loss of the precious mRNA. 

The many types of arrays/chips commercially available require different 

quantities of mRNA. The possibility of scaling the isolation protocols allows 

you to isolate mRNA from any quantity of cells. Dynabeads ® Oligo(dT) 25 can 

therefore be used to prepare the amount of mRNA required for any type of 

micro- and macroarrays or chips (one example is shown in fig. 8). Please 

contact Technical Support or go to www.dynalbiotech.com for a list of 

references where Dynabeads ® have been used to prepare mRNA for various 

types of analysis. 

mRNA in Automated Protocols and IVD Assays 

Sample preparation with Dynabeads ® Oligo(dT) 25 facilitates efficient mRNA 

concentration. No centrifugation or precipitation steps are required, as 

washing and buffer-changes are easily accomplished by the use of a magnet. 

The scalable magnetic mRNA isolation protocols are perfect for automation 

and can be implemented on most liquid handling robotic platforms (fig. 9). 

For further information on the available protocols, please contact Technical 

Support or visit our web-site at www.dynalbiotech.com. 

The unique uniformity of size, surface area and superparamagnetic properties 

of the Dynabeads ® are not merely for cosmetic benefit. These properties are 

also the basis for optimal accessibility and reaction kinetics, in turn enabling 

rapid, yet sensitive, and reliable capture of mRNA. The beads do not inhibit 

enzymatic activity, and bead-bound mRNA can be included in downstream 

magnetic handling as well as detection assays. 

The uniform characteristics and low batch-to-batch variation of the 

Dynabeads ® allow our customers to cut costs in their internal QC-testing, 

without compromising the reproducibility or quality of the results. 

ViroLogic, Inc. (San Francisco, CA, USA) uses Dynabeads ® Oligo(dT) 25 in 

their advancement in the fields of individualised medicine and pharmacogenomics. 

In addition to being a service company, ViroLogic, Inc. discovers 

and develops innovative products used by pharmaceutical companies to 

guide and improve treatment of serious viral diseases such as AIDS and 

hepatitis. Their fast and sensitive PhenoSense technology is used for 

assessing phenotypic drug resistance and susceptibility in viruses that cause 

these diseases. For general information on the availability of Dynabeads ® 

supplied on an OEM basis, please refer to chapter 7. See also chapter 11 for 

further information on Dynal Biotech’s Quality Systems. 

76 


Ordering Information 


References for mRNA Isolation and Applications 

77 

Relevant Dynabeads ® References 

1. Karrer EE. et al. (1995) In situ isolation of 

mRNA from individual plant cells: creation of 

cell-specific cDNA libraries. PNAS 92:3814- 

3818. 

2. Klein CA. et al. (2002) Combined transcriptome 

and genome analysis of single 

micrometastatic cells. Nature Biotech 

20:387-392. 

3. Konishi Y. et al. (2002) Isolation of living 

neurons from human elderly brains using 

the immunomagnetic sorting DNA-linker 

system. Am J Pathol 161:1567-1576. 

Product Volume Product No. 

Dynabeads ® Oligo(dT) 25 

Dynabeads ® mRNA DIRECT Kit* 

Dynabeads ® Oligo(dT) 25 , Lysis/Binding Buffer, Washing Buffers, 

10 mM Tris-HCl, Reconditioning Solution and Storage Buffer. 

Dynabeads ® mRNA DIRECT Micro Kit 

Dynabeads ® Oligo(dT) 25 , Lysis/Binding Buffer, Washing Buffers and 10 mM Tris-HCl. 

Dynabeads ® mRNA Purification Kit 

Dynabeads ® Oligo(dT) 25 , Binding Buffer, Washing Buffer and 10 mM Tris-HCl. 

* 5 times more Dynabeads ® Oligo(dT) 25 added. 

NEW 

2 ml 

5 ml 

50 ml 

20 isolations 

40 isolations 

610.02 

610.05 

610.50 

610.11 

610.12 

100 isolations 610.21 


6 

7 



Fig. 10. Principle for isolation of DNA using 

Dynabeads ® DNA DIRECT TM Universal. 

Fig. 11. Dynabeads ® DNA DIRECT TM are suitable 

for isolating pure, high quality DNA down to 10 

cells. DNA was isolated from different numbers 

of leucocytes, and used for PCR amplification of 

the GAPDH gene. 

Product Sample Volume 

Dynabeads ® DNA DIRECT Universal 

Dynabeads ® DNA DIRECT Blood 

Dynabeads ® Genomic DNA Blood 

Table 3: Which product to choose for your DNA isolation. 

Genomic DNA Isolation 

High quality DNA is crucial in DNA diagnostics, mutation detection and SNP 

analysis, pharmacogenomics, tissue typing and many other applications. 

Biomagnetic separation with Dynabeads ® offers a simple, rapid and easily 

automated method for isolating high purity DNA from a variety of different 

starting samples. 

Main Advantages of Dynabeads ® DNA Isolation: 

• PCR-ready DNA isolated in down to 10 minutes. 

• Eliminate contaminating inhibitors. 

• Highly reproducible results. 

• Perfect for automation. 

PCR-ready genomic DNA of high molecular weight can be isolated using 

Dynabeads ® . The protocols consist of only a few steps of pipetting and 

magnetic separation, with all operations performed in a single tube. 

Contaminating PCR inhibitors are eliminated without any centrifugation steps 

or use of phenol/chloroform. 


The Dynabeads ® DNA DIRECT Universal kit (Product no. 630.06) is 

designed for user-friendly isolation of genomic DNA directly from crude 

material in only 10 minutes. The kit is particularly useful for isolation of 

DNA from bacteria and cultured cells, and also from clinical specimens and 

tissues from various species. Only a minute amount of starting material is 

required. At least 200 ng of PCR-ready DNA is isolated per sample, sufficient 

for at least 10 PCR reactions. 

The Dynabeads ® DNA DIRECT Blood kit (Product no. 631.02) supports 

100 isolations from 100 µl of blood per sample, sufficient for at least 100 

PCR amplifications. The protocol can be modified to allow isolation from 

500 µl blood samples, providing enough template DNA for 1,000 PCR 

amplifications. 

The Dynabeads ® Genomic DNA Blood kit (Product no. 634.01/02) is designed 

for isolation of ultra-pure DNA from 200 µl fresh human blood. In the two-step 

procedure, leucocytes are first isolated from the erythrocytes and serum by 

both Dynabeads ® CD15 and CD45. DNA from lysed pure leucocytes is then 

captured onto DNA binding Dynabeads ® . 

Fresh/frozen blood ≤ 30 µl 

Cultured cells ≤ 10 4 cells 

Animal/plant tissue ≤ 100 mg 

Bacteria ≤ 10 7 -10 8 cells 

Fresh or frozen blood, 

bone marrow 

Fresh blood (stored up to 

4 days at 4-25 0 C) 

100-500 µl 

200 µl 

78 

Typical 

Applications 

PCR 

PCR, RFLP, SSCP, 

Southern Blotting 

Any downstream 

application 

Automated 

protocols 

TECAN Genesis ® 

Kingfisher ® 

Biomek ® 2000 

TECAN Genesis ® 

Kingfisher ® mL, Biomek ® FX 


A Variety of Sample Types and Downstream 

Applications 

DNA from Bacteria 

Dynabeads ® DNA DIRECT Universal are particularly useful for rapid isolation 

of bacterial genomic DNA from bacterial cultures, and also for isolating DNA 

from bacteria in food, clinical or environmental samples. 

Specific examples include: Listeria monocytogenes in milk, Leptospira 

interrogans in blood, cyanobacteria in water samples, Neisseria meningitidis, 

Haemophilus influenza, Streptococcus pneumoniae, Streptococcus agalactiae, 

Listeria monocytogenes and E. coli in cerebrospinal fluid and blood, (fig. 12) 

and Mycobacterium tuberculosis in lung and lymph node tissue. For a 

complete reference list, please visit www.dynalbiotech.com. 

DNA from Blood 

DNA isolation kits from Dynal Biotech are suitable for isolation of DNA from 

blood. 

Dynabeads ® DNA DIRECT Universal are recommended for the rapid isolation 

of DNA from small volumes, or even finger-prick samples, of blood. One test 

will isolate 600 ng - 1 µg DNA from a 30 µl blood sample, sufficient for 30-50 

PCR amplifications. The protocol can be fully automated on most liquid 

handling robots. 

The Dynabeads ® DNA DIRECT Blood kit isolates DNA from up to 0.5 ml 

blood samples in less than 15 minutes. One test provides enough template 

DNA for up to 1,000 PCR amplifications. The protocol is amenable to semiautomation 

as it includes only one centrifugation step. 

Dynabeads ® Genomic DNA Blood kit isolates DNA from 200 µl fresh blood 

and typically yields between 6-9 µg DNA per sample depending on the 

number of white blood cells in the blood sample. The protocol is ideal for 

walk-away automation. 

DNA from Other Clinical Samples 

Dynabeads ® DNA DIRECT Universal is excellent for the rapid isolation of DNA 

from small amounts of solid tumours, mouth wash (fig. 13), buccal scrapes, 

urine, bile and faeces. After DNA isolation, the beads can be added directly 

to the PCR without prior elution. The excellent sensitivity allows isolation of 

DNA even from single cells. 

DNA for Tissue Typing 

Dynal Biotech HLA Diagnostics offers a complete range of products for tissue 

typing. For more information see chapter 9 or go to www.dynalbiotech.com. 

The Dynabeads ® DNA DIRECT Blood kit is ideal for rapid and simple DNA 

extraction for low-resolution tissue typing with the Dynal Biotech Reli SSO 

products. The product isolates DNA from up to 0.5 ml blood, and the DNA is 

sufficient for at least 13 SSO PCR amplifications. 


79 

A Variety of Sample Types 

Fig. 12: PCR-products from DNA preparations using 

the Dynabeads ® DNA DIRECT Universal kit, from 

the following strains: N. meningitidis (lanes 2-3), 

H. influenzae (lanes 4-5), S. pneumoniae (lanes 

6-7), S. agalactiae (lanes 8-9), L. monocytogenes 

(lanes 10-11) and E. coli (lanes 12-13). Lanes 1 

and 16: MW markers; lane 14: negative control; 

lane 15: positive PCR-control. MW-sizes of all 

PCR-products were as expected. Courtesy of 

A. Bäckman, Örebro Medical Center Hospital, 

Örebro, Sweden. 

Fig. 13: PCR on DNA isolated from mouth wash 

specimen with Dynabeads ® DNA DIRECT TM . 

Lane 1: 100 bp ladder. Lane 3: extraction control 

(isolation from water). Lane 4: negative PCR 

control. Lanes 5-12: 157 bp K-ras PCR product 

from patient samples. Courtesy of M. Longfellow, 

Molecular Pathology Laboratory, The General 

Infirmary, Leeds, UK. 

6 

7 


A Variety of Downstream Applications 

Fig. 14: Identification of 3 mice transgenic 

for human Retinoic Acid Receptor (RAR) cDNA 

sequences. DNA isolated from 3-5 mg tail-tissue 

from each of 6 mice using the Dynabeads ® DNA 

DIRECT Universal kit. GAPDH amplification was 

used as a positive PCR control. Only 10% of the 

isolated DNA was used as template. 


DNA for the Identification of Transgenic Animals 

Dynabeads ® DNA DIRECT Universal kit is ideal for isolating DNA directly 

from small tissue samples. Mouse tail snips are often a preferred source of 

DNA for PCR-based identifications of transgenic animals in a litter (fig. 14). 

With Dynabeads ® DNA DIRECT Universal, PCR-ready DNA is isolated in 

less than 10 minutes. If desired, Dynabeads ® DNA DIRECT Universal can 

also be used to isolate DNA from mouse ear punch tissue, whole blood and 

other starting samples. 

Automated DNA Isolation Protocols 

The medium to high throughput Dynabeads ® DNA isolation protocols are 

implemented on a variety of liquid handling robotic platforms, thereby 

reducing the risk of human error while improving quality and reproducibility. 

Biomagnetic sample preparation allows for efficient target concentration 

without any centrifugation or precipitation steps. Washing and buffer changes 

are easily accomplished by the use of a magnet. 

For further information and to receive the scripts for the available automated 

DNA isolation protocols on any particular platform, please contact Dynal 

Biotech Technical Support. You can also download the scripts from our website 

at www.dynalbiotech.com. 

The low batch-to-batch variation of Dynabeads ® guarantees the quality and 

reproducibility of your results. For general information on the availability of 

Dynabeads ® supplied on an OEM basis, please refer to chapter 7. See also 

chapter 11 for further information on Dynal Biotech’s Quality Systems. 


Dynabeads ® DNA DIRECT Universal 

Dynabeads ® supplied in a Lysis Buffer, 10 M NaOH, 

Washing Buffer and Resuspension Buffer. 

Dynabeads ® DNA DIRECT Blood 

Red Cell Lysis Buffer, Dynabeads ® supplied in a Lysis Buffer, 

Washing Buffer and Resuspension Buffer. 

Dynabeads ® Genomic DNA Blood 

Dynabeads ® Pan Leucocytes, Cell Washing Buffer, BSA, 

Dynabeads ® supplied in a Lysis Buffer, Washing Buffer and Resuspension Buffer. 

80 



60 isolations 

384 isolations 

634.01 

634.02 


Dynabeads ® Streptavidin for Superior 

Results 

Dynabeads ® with recombinant streptavidin covalently attached to the bead- 

surface enable efficient isolation and handling of any biotinylated target 

molecule (fig. 15). The very high binding affinity of the streptavidin-biotin 

interaction (K D = 10 -15 ) is utilised in a vast number of molecular applications. 

Some of these applications are described on the following pages. 

Main Advantages of Dynabeads ® Streptavidin: 

• Flexible solid-phase protocols with liquid-phase reaction kinetics. 

• Direct and fast capture of any biotinylated target. 

• High batch-to-batch reproducibility gives consistent results in your 

application. 

• Biomagnetic protocols are easily adapted to automated platforms. 

The streptavidin-coupled Dynabeads ® are coated with a monolayer - not a 

multilayer - of recombinant streptavidin. This leaves the vast majority of the 

biotin binding sites sterically available for binding, not only of free biotin, 

but also for binding of biotinylated ligands/targets. 

The absence of excess physically adsorbed streptavidin ensures that only a 

negligible amount of streptavidin will be able to leak. Batch-to-batch variability 

is minimised, and the reproducibility of your results is optimised. 


Three different streptavidin-coupled Dynabeads ® are available to meet the 

requirements of different applications. Dynabeads ® M-270 Streptavidin and 

Dynabeads ® MyOne Streptavidin C1 are particularly suitable for nucleic acid 

applications (table 4). Their negatively charged hydrophilic surface (at pH 7) 

ensures negligible non-specific binding of irrelevant nucleic acids. Lysis in GTC 

buffers with the beads present is possible, and the beads also show minimal 

aggregation in high salt buffers used, for example, for hybridisation. 

Dynabeads ® M-270 Streptavidin (Product no. 653.05/06/07) 

2.8 µm medium to high capacity beads ideal for applications such as 

preparation of single-stranded DNA, SAGE, and solid-phase hybridisation 

(fig. 16). The production of Dynabeads ® M-270 Streptavidin follows a 

validated process in compliance with the cGMP for medical devices. 

Dynabeads ® MyOne Streptavidin C1 (Product no. 650.01/02/03) 

1.0 µm ultra-high capacity beads ideal for molecular displays and nucleic 

acid assays. The small size, combined with the characteristic shape, 

(fig. 17), gives a very high surface area. This means high capacity for the 

corresponding target molecule and maximum signal generation. The beads 

have a high iron content that ensures rapid magnetic separation. The 

uniform size, low sedimentation rate and high magnetic mobility together 

make these beads the optimal choice for any automated application. 

Dynabeads ® M-280 Streptavidin (Product no. 112.05/06, 602.10) 

2.8 µm beads suitable for a wide range of applications. Please see chapter 

7 for further information on this product. 


81 

Streptavidin-Coupled Dynabeads ® 

Mixed starting sample 

® 

Dynabeads Streptavidin 

Any biotinylated ligand 

Target binding 

Magnetic separation 

Fig. 15: Direct/indirect magnetic separation via 

the biotin–streptavidin interaction. A wide variety 

of ligands/probes can be used to isolate target 

molecules, relevant to your specific application 

or assay. 

Fig. 16: 2.8 µm Dynabeads ® 

Fig. 17: 1.0 µm Dynabeads ® 

6 

7 


Amount immobilised (pmol/mg beads) 


90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

55 

22 

35 

11 

67,5 

24,5 

100 1000 10000 

Fragment length (bp) 

83,4 

Fig. 18: B&W Buffer (red) is the standard buffer 

recommended for use with Dynabeads ® M-280 

Streptavidin. The specially designed Binding 

Solution (green) supplied in the Dynabeads ® 

kilobase-BINDER Kit significantly increases the 

binding capacity for large (>2kb) double stranded 

DNA fragments. 

® 

Dynabeads Streptavidin 

+ 

Immobilised DNA fragment 

Biotinylated DNA fragment 

Denature and remove non-biotinylated strand 

Fig. 19: Double-stranded PCR products immobilised 

onto Dynabeads ® Streptavidin are easily 

converted to single-stranded templates for use 

in downstream applications. Elution conditions 

can be heating, alkali denaturation or change in 

ionic strength. 

Dynabeads ® M-270 Streptavidin Dynabeads ® MyOne TM Streptavidin C1 

Basic bead Hydrophilic carboxylic acid Hydrophilic carboxylic acid 

Diameter 2.8 ± 0.2 µm 1.05 ± 0.1 µm 

Size distribution cv < 3% cv < 3% 

Blocking protein None None 

Isoelectric point pH 4.5 pH 5.2 

Charge at pH 7 - 50 mV - 35 mV 

Iron content (Ferrites) 14% (20%) 26% (37%) 

Free biotin binding > 650 - 1350 pmol/mg > 2500 pmol/mg 

Binding of biotinylated Ig 5 - 10 µg/mg 15 - 20 µg/mg 

Table 4: Overview of the characteristics for the two types of streptavidin-coupled Dynabeads ® recommended for nucleic acid applications. 

Selected Dynabeads ® Streptavidin 

Applications 

Over the last 15 years, streptavidin-coupled Dynabeads ® have been used in 

a large number of labs for a wide range of applications, several of which are 

covered by patents or patent-applications. 

The biotin-streptavidin linkage system is extremely flexible and can be used 

to capture almost any biotinylated target for a wide variety of downstream 

applications. On the following pages we present some of the key applications 

for which Dynabeads ® Streptavidin are used. 

Preparing Single-Stranded DNA Templates 

The immobilisation of a double-stranded PCR product for preparation of 

two single-stranded DNA templates is maybe the most widely used protocol 

for Dynabeads ® Streptavidin. A PCR product is first generated by using one 

biotinylated PCR primer, then the PCR product is immobilised onto the beads 

for convenient separation of the biotinylated and non-biotinylated strands 

using a magnet (fig. 19). 

The streptavidin-coupled Dynabeads ® will not inhibit enzymatic activity. This 

enables further handling and manipulation of the bead-bound DNA to be 

performed directly on the solid-phase. 

Both the immobilised and the eluted single-stranded DNA template can 

be used in downstream applications such as MALDI-MS, pyrosequencing 

technology and SNP analysis, as well as single-strand conformation 

polymorphism (SSCP), solid-phase DNA sequencing, DNA chips, primer 

extension and other molecular techniques. 

82 


Isolating RNA and DNA Binding Proteins 

Certain DNA and RNA binding proteins have been shown to be associated 

with specific genetic diseases. These often short-lived and low abundance 

regulatory proteins are considered to be potential drug targets. 

Sequence-specific DNA/RNA binding proteins are rapidly and efficiently 

isolated using Dynabeads ® Streptavidin, providing a superior and wellestablished 

alternative to affinity chromatography methods (fig. 20). The 

biotin labelled DNA/RNA sequence of interest is immobilised onto the beads 

and incubated with the cell extract. The proteins that bind to the sequence 

are isolated using magnetic separation. The protein can then be eluted off 

and characterised, or the bead-DNA/RNA-protein complexes can be applied 

directly in DNase footprinting studies. 

Dynabeads ® Streptavidin are recommended for this application, due to the 

very low non-specific binding of proteins to these beads. As described below, 

the dedicated Dynabeads ® kilobaseBINDER Kit is recommended for the 

immobilisation of larger DNA fragments. 

Immobilising Large DNA Fragments 

The amount of biotinylated DNA immobilised to Dynabeads ® Streptavidin 

will depend on fragment size. Due to steric hindrance, the binding efficiency 

is significantly reduced when the fragment size exceeds 2 kb. The specially 

designed Dynabeads ® kilobaseBINDER Kit (Product no. 601.01) includes 

a patented immobilisation activator, significantly increasing the binding 

efficiency of large dsDNA fragments (fig. 18). 

This product has proven useful for innovative applications in cell biology, 

including: chromatin beads mimicking chromosomes for studying selforganisation 

of microtubules into bipolar spindles (fig. 21), synthetic nuclei 

for studying RNA export from the nucleus in vitro, reconstitution of nuclear 

movement along microtubules and examination of the molecular basis of this 

movement in vitro. 

A reference list is available upon request. 


83 

A Wide Variety of Applications 

Fig. 20: Comparison of C2 murine skeletal 

myotube extract and purified MS2, a single DNA 

binding protein of 43 kDa. Denaturing 12% SDS- 

PAGE gel. Lane 1: 5 ul pre-stained marker proteins, 

lane 2: 100 ug C2 cell extract, lane 3: 0.5 µg MS2 

isolated from C2 cell extract using Dynabeads ® 

M-280 Streptavidin and a double-stranded DNA 

sequence. Courtesy of L. Ren, H. Cheng and 

E.A. Sternberg, Medical College of Wisconsin 

Milwaukee, WI, USA. 

Fig. 21: DNA-coated Dynabeads ® (red) assemble 

a mitotic spindle (green) indistinguishable 

from that in a normal dividing cell (1). Plasmid 

DNA bound to the beads using the Dynabeads ® 

kilobaseBINDER Kit generates a well-defined 

source of chromatin for studying spindle assembly 

in Xenopus egg extracts. Courtesy of R. Heald, 

E. Karsenti, A. Hyman, EMBL, Heidelberg, 

Germany. 

6 

7 


A Wide Variety of Applications 

Fig. 22. Isolation of tRNA-VAL from rat mitochondria. 

S: supernatant after capture, W: washing 

solution, E: eluate, position of the isolated 

full-length tRNA-VAL is indicated by the arrow. 

Courtesy of M. Mørl, University of Munich, 

Germany. 


Specific Capture of Nucleic Acids 

Double-stranded DNA fragments immobilised to Dynabeads ® Streptavidin 

are easily converted to single-stranded DNA templates or probes (fig. 19). 

The bead-bound sequences can then be used for solid-phase hybridisation 

capture of specific DNA/RNA sequences (fig. 22). 

Streptavidin-coupled Dynabeads ® provide an efficient solid-phase alternative 

to nitrocellulose. An inherent benefit of magnetic handling is that downstream 

manipulations and buffer changes can be done by simply concentrating the 

bead-bound target at the tube-wall by using a magnet. The excellent reaction 

kinetics of the Dynabeads ® is comparable to liquid phase kinetics. A singlestranded 

biotinylated probe can be bound directly to the beads for capture of 

target sequences. Alternatively, an indirect capture approach can be used, as 

the reaction kinetics are faster, in some cases, when the biotinylated probe 

is added to the sample for target hybridisation, prior to immobilisation to the 

beads. The 1.0 µm Dynabeads ® MyOne Streptavidin C1 have a very high 

surface area per mg of beads, enabling high enrichment of low abundance 

DNA/RNA. The slightly negatively charged surfaces of both the Dynabeads ® 

M-270 Streptavidin and the Dynabeads ® MyOne Streptavidin C1 also 

ensure negligible non-specific binding of non-target DNA sequences. 

Examples of applications include; sequence-specific capture, cDNA selection 

and enrichment, isolation of cell-specific transcripts and mRNA differential 

display. 

A reference list is available upon request. 


Dynabeads ® MyOne Streptavidin C1 

Dynabeads ® M-270 Streptavidin 

Dynabeads ® M-280 Streptavidin 

Dynabeads ® kilobaseBINDER 

Dynabeads ® M-280 Streptavidin, Binding Solution and Washing Solution. 

Please see chapter 7 for additional Dynabeads ® Streptavidin products. 

84 

2 ml (10 mg/ml) 

10 ml (10 mg/ml) 

100 ml (10 mg/ml) 

2 ml (10 mg/ml) 

10 ml (10 mg/ml) 

100 ml (10 mg/ml) 

2 ml (10 mg/ml) 

10 ml (10 mg/ml) 

100 ml (10 mg/ml) 

650.01 

650.02 

650.03 

653.05 

653.06 

653.07 

112.05 

112.06 

602.10 



Dye Terminator Removal 

Removal of unincorporated dye terminators, salts and other unwanted 

components from the extension products is critical for both standard and 

capillary DNA sequencing systems. Dynabeads ® Sequencing Clean-Up is a 

simple three-step magnetic bead based system for purifying DNA sequencing 

reactions (fig. 23). The unique uniformity of the beads guarantees consistently 

high quality, reproducible results, long reading lengths and high Phred20 

scores (fig. 24 and 25). 

Dynabeads ® Sequencing Clean-Up removes dye terminators from 10-20 µl 

sequencing reactions and is compatible with any sequencing chemistries 

including including BigDye, dRhodamine dye, Rhodamine, DYEnamic 

ET and WellRED terminators. After purification, the extension products can 

be separated on any DNA sequencing instrumentation. 

Main Advantages of Dynabeads ® Sequencing Clean-Up: 

• Efficient removal of excess dye terminators. 

• Fast 15 minutes procedure. 

• No centrifugation or filtration. 

• Automated protocols are available (see www.dynalbiotech.com). 



85 

Sequencing Clean-up 

Fig. 23. Principle for purification of DNA 

sequencing preparation using Dynabeads ® 

Sequencing Clean-up. 

Fig. 24. BAC-clone 1105B13 directly sequenced 

using BigDye ® Terminator Cycle Sequencing 

Ready Reaction Kit (ABI). The sequencing products 

were purified using the sample injected into 

an ABI PRISM ® 310 automated capillary DNA 

sequencer. 

Fig. 25. M13 mp18 + template sequenced in 

a reaction using DYEnamic TM ET, purified with 

Dynabeads ® Sequencing Clean-Up and analysed 

on the MegaBase TM 1000. 


Dynabeads ® Sequencing Clean-Up 500 samples 661.01 

Dynabeads ® Sequencing Clean-Up 2500 samples 661.02 

6 

7 


86

Nucleic Acid Isolation.indd - Invitrogen

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