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HHuummaann GGeennoommiicc DDNNAA

IIssoollaattiioonn MMeetthhooddss

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IInnttrroodduuccttiioonn

Double-stranded DNA is remarkably inert chemical. Its potentially reactive groups are

buried within the central helix, tied up in hydrogen bonds. Its base pairs are protected on the

outside by a formidable casing of phosphates and sugars and are reinforced internally by

strong stacking forces. With such robust shielding and scaffolding, DNA outlasts most other

intracellular components in locations as disparate as modern day crime scenes and ancient

burial sites. The same chemical durability endows libraries of genomic DNA with both

permanence and value, thereby enabling genetic engineering and sequencing projects, both

large and small.

Despite its chemical stability, double-stranded DNA is nevertheless physically

fragile. Long and snaky, with little lateral stability, high-molecular-weight DNA is

vulnerable to hydrodynamic shearing forces of the most modest kind. Double-stranded DNA

behaves in solution as a random coil that is stiffened by stacking interactions between the

base pairs and electrostatic repulsion between the charged phosphate groups in the DNA

backbone. Hydrodynamic flow—resulting from pipetting, shaking, or stirring – generates

drag on the stiffened coil and has the capacity to shear both strands of the DNA. The longer

the DNA molecule, the weaker the force required for breakage. Genomic DNA is therefore

easy to obtain in the fragmented form but becomes progressively more difficult to isolate as

the desired molecular weight increases. DNA molecule >150 kb are prone to breakage by

forces generated during procedures commonly used to isolate genomic DNA.

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DDNNAA EExxttrraaccttiioonn

DNA Extraction is the removal of deoxyribonucleic acid (DNA) from the cells or viruses in

which it normally resides.

AApppplliiccaattiioonnss

Extraction of DNA is often an early step in many diagnostic processes used to detect

bacteria and viruses in the environment as well as diagnosing disease and genetic disorders.

DNA is suitable for SSR analysis and other PCR-based applications.

These techniques include but are not limited to --

Fluorescence In Situ Hybridization (FISH): FISH is a molecular technique that is

used, among other things, to identify and enumerate specific bacterial groups.

Terminal Restriction Fragment Length Polymorphism (T-RFLP): T-RFLP is

used to identify, characterize, and quantify spatial and temporal patterns in marine

bacterioplankton communities.

Sequencing: Portions of, or whole genomes may be sequenced as well as extra

chromosomal elements for comparison with existing sequence in the public data base.

DDNNAA IIssoollaattiioonn TTeecchhnniiqquueess

Many different methods and technologies are available for the isolation of genomic

DNA. In general, all methods involve disruption and lysis of the starting material followed

by the removal of proteins and other contaminants and finally recovery of the DNA.

Removal of proteins is typically achieved by digestion with proteinase K, followed by

salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-

exchange or silica technology). DNA is usually recovered by precipitation using ethanol or

isopropanol. The choice of a method depends on many factors: the required quantity and

molecular weight of the DNA, the purity required for downstream applications, and the time

and expense.

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The separation of DNA from cellular components can be divided into four stages:

1. Disruption

2. Lysis

3. Removal of proteins and contaminants

4. Recovery of DNA

HHuummaann GGeennoommiicc DDNNAA IIssoollaattiioonn MMeetthhooddss

Several methods for extraction of genomic DNA from blood, tissue, sperm, tooth and bone

have been examined and demonstrated so far. Some of these are following:

1. Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K

and Phenol

2. Isolation of High-molecular-weight DNA from Mammalian Cells Using Formamide

3. Isolation of DNA from Mammalian Cells by spooling

4. Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates

5. Rapid isolatin of Mammalian DNA

IIssoollaattiioonn ooff HHiigghh--mmoolleeccuullaarr--wweeiigghhtt DDNNAA ffrroomm MMaammmmaalliiaann

CCeellllss UUssiinngg PPrrootteeiinnaassee KK aanndd PPhheennooll

This procedure is derived from a method originally described by Daryl Stafford and

colleagues (Blin and Stafford 1976).It is the method of choice when large amounts of

mammalian DNA are required, for example, for Southern blotting or for construction of

genomic libraries in bacteriophage λ vectors.

Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x

10⁷ culture aneuploid cells (e.g., Hela cells). The usual yield of DNA from 20 ml of normal

blood is ~ 250 µg.

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IIssoollaattiioonn ooff HHiigghh--mmoolleeccuullaarr--wweeiigghhtt DDNNAA ffrroomm

MMaammmmaalliiaann CCeellllss UUssiinngg FFoorrmmaammiiddee

This protocol is a modification of the procedure of Kupiec et al. (1987) and involves

digestion of cells and tissues with proteinase K, dissociation of DNA-protein complexes

(chromatin) with high concentrations of formamide, and removal of the protease and organic

solvent by extensive dialysis through collodion bags.

Formamide is an ionizing solvent that both dissociates protein-DNA complexes and,

subsequently, denatures the released proteins. However, it does not significantly affect the

activity of proteinase K.

The genomic DNA prepared by this procedure is large (>200 kb) and suitable for the

construction of libraries in high-capacity vectors and for the analysis of large DNA

fragments by pulsed-field gel electrophoresis.

DDiissaaddvvaannttaaggeess

The method has two disadvantages:

1. It requires more time than other procedures.

2. The concentration of DNA in the final preparation is low (~10 µg/ml)

Approximately 1 mg of high-molecular-weight DNA can be prepared from 1 x 10⁸

cultured aneuploid mammalian cells (e.g., Hela cells).

IIssoollaattiioonn ooff DDNNAA ffrroomm MMaammmmaalliiaann CCeellllss bbyy ssppoooolliinngg

This method, adapted from Bowtell (1987), is used to prepare DNA simultaneously

from many different samples of cells or tissues. The key steps in the protocol are (1)

precipitation of the genomic DNA at the interface between the cell lysate and a layer of

ethanol, followed by (2) spooling of the precipitated DNA onto a Shepherd’s crook. The

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DNA is then lifted from the ethanolic solution on the crook and dissolved in the aqueous

buffer of choice.

This method of collecting precipitates of high-molecular-weight DNA was first used in

the 1930s. Small fragments of DNA and RNA are not efficiently incorporated into the

gelatinous spool. Although the DNA is generally too small (~80 kb) for efficient

construction of genomic DNA libraries, it gives excellent results in southern hybridizations

and polymerase chain reactions and can be used to construct a size-fractionated library after

limited digestion with a restriction enzyme.

Cultured aneuploid mammalian cells (2.0 x 10⁷, e.g., HeLa cells) yield 100 µg of DNA

in a volume of ~1 ml.

IIssoollaattiioonn ooff DDNNAA ffrroomm MMaammmmaalliiaann CCeellllss GGrroowwnn iinn

9966--wweellll MMiiccrroottiitteerr PPllaatteess

This protocol is adapted from Ramirez-Solis et al. It describes a simple and efficient

method for extracting genomic DNA from eukaryotic cells grown in the individual wells of

microtiter plates.

Each well yields sufficient genomic DNA for several standard polymerase chain

reactions (PCRs) or for analysis in a single lane of a Southern hybridization.

RRaappiidd IIssoollaattiinn ooff MMaammmmaalliiaann DDNNAA

Mammalian DNA prepared according to this protocol is 20-50 kb in size and suitable

for use as a template in PCRs. The yields of DNA vary between 0.5 and 3.0 µg/mg tissue

and 5 and 15 µg per 300 µl of whole blood.

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EExxttrraaccttiioonn ooff HHuummaann nnuucclleeaarr DDNNAA ffrroomm BBlloooodd

UUssiinngg PPrrootteeiinnaassee KK aanndd PPhheennooll

PPrriinncciippllee

This method involves digesting eukaryotic cells or tissues with proteinase K in the

presence of EDTA and solubilizing membranes and denaturing proteins with a detergent

such as SDS. The nucleic acids are then purified by phase extractions with organic solvents.

Contaminating RNA is eliminated by digestion with an RNase, and low-molecular-weight

substances are removed by dialysis.

This method can be scaled to yield amounts of DNA ranging from less than ten to more

than hundreds of micrograms of DNA. However, shearing forces are generated at every step,

with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in

length.

DNA of this size is adequate for Southern analysis on standard agarose gels, as a

template in polymerase chain reactions (PCRs), and for the construction of genomic DNA

libraries in bacteriophage λ vectors.

The quality of DNA extracted from liquid blood is not adversely affected by storage at

4ºC for up to 24 h. Small but significant changes have been observed in metabonomic studies

in samples of blood maintained at 48°C for 36 h.

MMaatteerriiaallss

BBuuffffeerrss aanndd ssoolluuttiioonnss

1. Ammonium acetate (10 M)

2. Dialysis buffer

50 mM tris-cl (p.H 8.0)

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10 mM EDTA (p.H 8.0)

3. Ethanol

4. Lysis buffer

10 mM Tris-cl (p.H 8.0)

mM EDTA (p.H 8.0)

0.5 % (w/v) SDS

20 µg/ml DNase-free pancreatic RNase

The first three ingredients of the lysis buffer may be mixed in advance and stored at room

temperature. RNase is added to an appropriate amount of the mixture just before use.

Adding RNase to the lysis buffer eliminates the need to remove RNA from semi purified

DNA at a later stage in the precipitation.

5 Phenol, equilibrated with 0.5 M Tris-cl (p.H 8.0)

6 TE (p.H 8.0)

7 Tris-buffered saline (TBS)

8 Acid citrate dextrose solution B (ACD)

0.48% w/v citric acid

1.32% w/v sodium citrate

1.47 % w/v glucose

9 EDTA

10 Phosphate buffered saline (PBS)

EEnnzzyymmeess aanndd bbuuffffeerr

Proteinase k (20 mg/ml)

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CCeennttrriiffuuggeess aanndd rroottoorrss

Sorvall centrifuge with H1000B and SS-34 rotors (or their equivalents)

EEqquuiippmmeennttss

Cut-off yellow tips

Dialysis tubing clips

Rocking platform or dialysis tubing

Spectrophotometer

Tube mixer or roller apparatus

Vacuum aspirator equipped with traps

Water bath

Wide-bore pipettes (0.3-cm diameter orifice )

SSaammppllee

Blood sample

PPrroocceedduurree

CCoolllleeccttiioonn ooff BBlloooodd

Collect cells from freshly drawn or frozen samples. Human blood must be collected under

sterile conditions.

TToo ccoolllleecctt cceellllss ffrroomm ffrreesshhllyy ddrraawwnn bblloooodd

I. Collect ~20 ml of fresh blood in tubes containing 3.5 ml of either acid citrate dextrose

solution B (ACD) or EDTA.

II. Transfer the blood to a centrifuge tube and centrifuge at 1300g for 15 minutes at 4º C.

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III. Remove the supernatant fluid by aspiration. Use a pasteur pipette to transfer the buffy

coat carefully to the fresh tube and repeat the centrifugation. Discard the pellet of red

cell. The buffy coat is a broad band of white blood cells of heterogeneous density.

IV. Remove residual supernatant from the buffy coat by aspiration. Resuspend the buffy

coat in 15 ml of lysis buffer. Incubate the solution for one hour at 37º C and proceed

to step 1.

TToo ccoolllleecctt cceellllss ffrroomm ffrroozzeenn bblloooodd ssaammpplleess

I. Collect 20 ml of fresh blood in tubes containing 3.5 ml of either acid citrate dextrose

solution B (ACD) or EDTA. The blood may be stored for several day at 0 C or

indefinitely at 70 C before the DNA is prepared.

II. Thaw the blood in water bath at room temperature and then transfer it to centrifuge

tube add an equal volume of phosphate buffered saline at room temperature.

III. Centrifuge the blood at 3500g for 15 minutes at room temperature.

IV. Remove the supernatant which contains lysed red cells, by aspiration. Resuspend the

pellet in 15 ml of lysis buffer. Incubate the solution for 1 hour at 37º C, and then

proceed to step 1.

TTrreeaattmmeenntt ooff LLyyssaattee wwiitthh PPrrootteeiinnaassee KK aanndd PPhheennooll

1. Transfer the Lysate to one or more Centrifuge tubes that fit into Sorvall SS-34 rotor,

or equivalent. The tube should not be more than one-third full.

2. Add Proteinase K (20 mg/ml) to a final concentration of 100 µg/ml. Use a glass rod to

mix the enzyme solution gently into the viscous lysate of cells.

3. Incubate the lysate in a water bath for 3 hours at 50º C. Swirl the viscous solution

from time to time.

4. Cool the solution to room temperature and add an equal volume of phenol

equilibrated with 0.1 M Tris-Cl (pH 8.0). Gently mix the two phases by slowly

turning the tube end-over-end for 10 minutes on a tube mixture. If the two phases

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have not formed an emulsion at this stage, place the tube on a roller apparatus for 1

hour.

5. Separate the two phases by centrifugation at 5000g (6500 rpm in a Sorvall SS-34

rotor) for 15 minutes at room temperature).

6. Use a wide –bore pipette (0.3-cm diameter orifice) to transfer the viscous aqueous

phase to a fresh centrifuge tube.

7. Repeat the extraction with phenol twice more and pool the aqueous phases.

IIssoollaattiioonn ooff DDNNAA

8. After the third extraction with phenol, transfer the pooled aqueous phases to a fresh

centrifuge tube and add 0.2 V of 10 M ammonium acetate. Add 2 vol of ethanol at

room temperature and swirl the tube until the solution is thoroughly mixed.

9. The DNA immediately forms a precipitate. Remove the precipitate in one piece from

the ethanolic solution with a Pasteur pipette. Contaminating oligonucleotides remain

in the ethanolic phase.

10. If the DNA precipitate becomes fragmented, collect the precipitate by centrifugation

at 500g (6500 rpm in a Sorvall SS-34) for 5 minutes at room tempeture.

11. Wash the DNA precipitate twice with 70% ethanol, and collect the DNA by

centrifugation.

12. Remove as much of 70% ethanol as possible, using an aspirator. Store the pellet of

DNA in an open tube at room temperature until the last visible traces of ethanol have

evaporated.

13. Don’t allow the pellet of DNA to dry completely; desiccated DNA is very difficult to

dissolve.

14. Add 1 ml of TE (Ph8.0) for each 0.1 ml of cells (step 1). Place the tube on a rocking

platform and gently rock the solution for 12-24 hours at 4º C until the DNA has

completely dissolved. Store the DNA solution at 4º C.

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MMeeaassuurree tthhee ccoonncceennttrraattiioonn ooff DDNNAA

15. A large sample (10-20µl) is withdrawn with an automatic pipetter equipped with cut-

off yellow tips. The sample is then diluted with ~0.5ml of TE (p.H 8.0) and vortexed

vigorously for 1-2 minutes. The absorbance of the diluted sample can be read at 260,

270 and 280 nm. A solution with an Absorbance of 1 at 260 contains ~50 µg of

DNA/ml.

More accurate measurement of DNA concentration can be made by fluorometry in the

presence of fluorescent dyes.

AAnnaallyyzzee tthhee ccoonncceennttrraattiioonn ooff DDNNAA

16. Analyze the quality of the preparation of high molecular weight DNA by pulsed-field

gel electrophoresis or by electrophoresis through a conventional 0.6 % agarose gel.

PPrreeccaauuttiioonnss && LLiimmiittaattiioonnss

1. DNA has been extracted from leucocytes and on prolonged storage of whole blood at

-20 and -80ºC, DNA yield was considerably decreased which was probably due to

degeneration of the white blood cells in the storage of long period.

2. Blood should not be collected into heparin, which is an inhibitor of the polymerase

chain reaction.

3. Tris may be harmful by inhalation, ingestion or skin absorption. Wear appropriate

gloves and safety glasses.

4. The pH of the phenol must be ~ 8.0 to prevent DNA from becoming trapped at the

interface between the organic and aqueous phases.

5. When transferring the aqueous phase, it is essential to draw the DNA into pipette very

slowly to avoid disturbing the material at the interface and to minimize hydrodynamic

shearing forces.

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6. Do not allow the pellet of DNA to dry completely; desiccated DNA is very difficult to

dissolve.

7. Do not be concerned if some of the DNA remains in the well, since DNA molecules >

250 kb have difficulty entering the gel.

RReessuulltt

The quality and quantity of extracted genomic DNA were controlled. High quality DNA was

obtained using our method. Blood samples stored at 4ºC for one year was able to profiling

for SSR and other PCR applications. Agarose gel 0.6% was used for quality control of

genomic DNA.