Nucleic Acid Isolation · 2017-10-19 · 71 7 6 Nucleic Acid Isolation Isolation of Pure, Intact...

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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

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

www.dynalbiotech.com70

Application Overview

Positive Isolation

Negative Isolation

Depletion

Expansion

DNA Isolation

Specific Protein IsolationmRNA Isolation

Organelle Isolation

Capture of Biotinylated Target

Samples:Whole BloodCord BloodMononuclear CellsBone MarrowBuffy CoatSpleenLymph NodeTissue Digest


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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.


mRNA Isolation

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

Fig. 1: Isolation of mRNA from different starting samples and for a wide variety of downstream applications.

TTTTTTTTTTTT

TTTTTTTTTTTT

TTTTTTTTTTTT

Crude lysatecontainingpoly A RNA

Animal / Plant tissue

Cultured cells Total RNA

Serum / Plasma

Reversetranscription

TTTTTTTTTTTTAAAAAAAAAAAA mRNA

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

Elute(optional)

TTTTTTTTTTTT+

AAAAAAAAAAAA mRNA


Regenerateand reuse


······· c

Northern blottingDot/blot hybridisationPrimer extension

translationS1 nuclease assayHybridisation probesDNA array/chip

In vitro

·······

RT-PCRcDNA synthesisSolid-phase cDNA librarySAGE-experimentsRACE-experimentsSubtractive hybridisationDifferential display

Pure cell populationisolated with Dynabeads®

+

Product Prod. No. Starting sample Sample size

Dynabeads® Oligo(dT)25

610.02610.05610.50

Any cells, animal- or plant tissue Scalable protocols

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

Dynabeads® mRNA DIRECT™ Kit610.11610.12

Any cells, animal- or plant tissue≤ 2 x 107 cells≤ 200 mg animal tissue≤ 400 mg plant tissue

Dynabeads® mRNA DIRECT™ Micro Kit 610.21 Any cells, animal- or plant tissue1-2.5 x 104 cells ≤ 5 mg animal tissue≤ 10 mg plant tissue


Isolate mRNA from Any Sample

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.

Species Starting Sample - Specific Tissue or Cell Type

Mammals

Bovine Corpus luteum

Dog Leucocytes

Gerbil Cochlear cells

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

Human

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

MouseBrain, 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

Pig Adipose tissue, Heart

RatAdrenals, 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

Birds Pigeon Breast muscle, Frozen tissues, Liver

FishTrout 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

Arabidopsis thalianaFlowers, Leaves, Roots, Rosette leaves, Seed, Silique, Stem, Stem leaves, Whole plants

Hordeum vulgareEmbryo, Endosperm , Leaves, Root, Seed aleurone, Seed embryos, Seed endosperm, Seed

Brassica oleracea Leaves, Root, Stigma

Lupinus angustifolius Cotyledon

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

YeastsCandida tropicalis Cell culture

Hansenula polymorpha Soil samples

Saccharomyces cerevisiae Cell culture, Soil samples

ParasitesCryptosporidium parvum Oocyst

Schistosoma mansoni Whole worm

Viruses

HCMV Infected cells

HIV-1Cells in broncho-alveolar washes, Cerebrospinal fluid, PBMC, Plasma, Serum, T lymphocytes cell line, CD4+ cells

HIV-2 Plasma

HTLV-I/II Infected cells

Human coronavirus Infected cells

Poliovirus Infected cells


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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).

Choose the Right Product

Fig. 2: The true uniform size and shape of Dynabeads® Oligo (dT)

25 provide rapid and

efficient binding kinetics and highly reproducible results.

New &

improved


mRNA from Pure Cell PopulationsMolecular 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 CellsIt 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).

A Wide Variety of mRNA Applications

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.

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 solid-phase 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).

Patient 1 Patient 2 Patient 3 Patient 4

+

MW

.

.

A

Patient 1 Patient 2 Patient 3 Patient 4

+

MW

.

.

B

Fig. 3: Positive isolation of a specific cell type using antibody-coated Dynabeads® , followed by mRNA isolation using Dynabeads® Oligo(dT)

25.

CK19

EPCAM

Number of cells: 1 1 5 5 10 10


Isolation of mRNA with Dynabead Oligo(dT)25sfor use in further downstream manipulations

Ò

Pure cells isolated with cell-specific DynabeadsÒ

AAAAAAAAATTTTTTTT


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Isolate mRNA bymagnetic separation

Synthesize first-strandcDNA without eluting themRNA off the Dynabeads®



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

TTTTTTTTTTTT

TTTTTTTTTTTT

TTTTTTTTTTTT

mRNA for cDNA LibrariesDynabeads® 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 PCRDynabeads® 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.


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 BlottingOnly 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.


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 time-course 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.


mRNA for MicroarraysDynabeads® 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 AssaysSample 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 pharmaco-genomics. 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.


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.

A B


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Relevant Dynabeads® References1. 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 transcrip-tome 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.

References for mRNA Isolation and Applications

Ordering Information

Product Volume Product No.


2 ml5 ml

50 ml

610.02610.05610.50

Dynabeads® mRNA DIRECT™ Kit* Dynabeads® Oligo(dT)

25, Lysis/Binding Buffer, Washing Buffers,

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

20 isolations40 isolations

610.11610.12

Dynabeads® mRNA DIRECT™ Micro Kit Dynabeads® Oligo(dT)

25, Lysis/Binding Buffer, Washing Buffers and 10 mM Tris-HCl.

100 isolations 610.21

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


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.

Choosing the Right Product

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®.


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

Product Sample VolumeTypicalApplications

Automatedprotocols

Dynabeads® DNA DIRECT™ Universal

Fresh/frozen blood ≤ 30 µl

PCR

TECAN Genesis®

Kingfisher® Biomek® 2000

Cultured cells ≤ 104 cells

Animal/plant tissue ≤ 100 mg

Bacteria ≤ 107-108 cells

Dynabeads® DNA DIRECT™ BloodFresh or frozen blood, bone marrow

100-500 µlPCR, RFLP, SSCP,Southern Blotting

Dynabeads® Genomic DNA Blood Fresh blood (stored up to 4 days at 4-250C)

200 µlAny downstream application

TECAN Genesis®

Kingfisher® mL, Biomek® FX

Fig. 11. Dynabeads® DNA DIRECTTM 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.

Fig. 10. Principle for isolation of DNA using Dynabeads® DNA DIRECTTM Universal.


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A Variety of Sample Types and Downstream Applications

DNA from BacteriaDynabeads® 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 BloodDNA 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 semi-automation 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 SamplesDynabeads® 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 TypingDynal 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.

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 DIRECTTM. 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.

A Variety of Sample Types


DNA for the Identification of Transgenic AnimalsDynabeads® 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 ProtocolsThe 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 web-site 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.

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.

A Variety of Downstream Applications



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 BloodDynabeads® Pan Leucocytes, Cell Washing Buffer, BSA,Dynabeads® supplied in a Lysis Buffer, Washing Buffer and Resuspension Buffer.

60 isolations384 isolations

634.01634.02


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Dynabeads® Streptavidin for Superior ResultsDynabeads® 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.

Choosing the Right ProductThree 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.

Mixed starting sample

Dynabeads Streptavidin®

Any biotinylated ligand

Magnetic separation

Target binding

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.

Streptavidin-Coupled Dynabeads®

Fig. 16: 2.8 µm Dynabeads®

Fig. 17: 1.0 µm Dynabeads®


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 TemplatesThe 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.

0

10

20

30

40

50

60

70

8090

100 1000 10000

22

55

11

Amou

nt immob

ilised

(pm

ol/m

g be

ads )

35

24,5

67,5

83,4

Fragment length (bp)

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.


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.

Biotinylated DNA fragmentDynabeads Streptavidin®

+

Immobilised DNA fragment

Denature and remove non-biotinylated strand

Dynabeads® M-270 Streptavidin Dynabeads® MyOneTM 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.


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Isolating RNA and DNA Binding ProteinsCertain 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 well-established 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 FragmentsThe 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 self-organisation 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.

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.

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.

A Wide Variety of Applications


Specific Capture of Nucleic AcidsDouble-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 single-stranded 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.

Fig. 22. Isolation of tRNA-VAL from rat mitochon-dria. 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.

A Wide Variety of Applications



Dynabeads® MyOne™ Streptavidin C1 2 ml (10 mg/ml)10 ml (10 mg/ml)

100 ml (10 mg/ml)

650.01650.02650.03

Dynabeads® M-270 Streptavidin 2 ml (10 mg/ml) 10 ml (10 mg/ml)

100 ml (10 mg/ml)

653.05653.06653.07

Dynabeads® M-280 Streptavidin 2 ml (10 mg/ml)

10 ml (10 mg/ml)100 ml (10 mg/ml)

112.05112.06602.10

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


Please see chapter 7 for additional Dynabeads® Streptavidin products.


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Sequencing Clean-up

Fig. 23. Principle for purification of DNA sequencing preparation using Dynabeads® Sequencing Clean-up.

Fig. 25. M13 mp18 + template sequenced in a reaction using DYEnamicTM ET, purified with Dynabeads® Sequencing Clean-Up and analysed on the MegaBaseTM 1000.

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.

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).

Dye Terminator Removal



Dynabeads® Sequencing Clean-Up 500 samples 661.01

Dynabeads® Sequencing Clean-Up 2500 samples 661.02

Nucleic Acid Isolation · 2017-10-19 · 71 7 6 Nucleic Acid Isolation Isolation of Pure, Intact...

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Transcript of Nucleic Acid Isolation · 2017-10-19 · 71 7 6 Nucleic Acid Isolation Isolation of Pure, Intact...