BCB 304

Recombinant DNA technology

Spring 2020

Lecture 2
Extraction and purification of nucleic acid
Preparation of total DNA

• Bacterial cells
• Human sperm cells
• Viral DNA
 Preparation of total DNA
 Preparation of total cell DNA from bacterial cells is divided
into four stages:

a) A culture of bacteria is grown and then harvested

b) The cells are broken open to release their contents
c) This cell extract is treated to remove all components
except the DNA
d) The resulting DNA solution is concentrated
Preparation of a cell extract
 Purification of DNA from a cell extract
 A variety of methods can be used to purify the DNA from this
mixture
a) Removing contaminants by organic extraction and enzyme
digestion
b) Using ion-exchange chromatography to purify DNA from a
cell extract (solid phase)

Non-organic extraction of DNA

 Purification of DNA from a cell extract
 Organic extraction and enzyme digestion
 The standard way to deproteinize a cell extract is to add phenol or a 1: 1
mixture of phenol and chloroform
 The organic solvents precipitate proteins but leave the nucleic acids (DNA
and RNA) in aqueous solution
 The cell extract is mixed gently with the solvent, and the layers then
separated by centrifugation, precipitated protein molecules are left as a
white coagulated mass at the interface between the aqueous and organic
layers
 The aqueous solution of nucleic acids can then be removed with a pipette
 Purification of DNA from a cell extract
 Organic extraction and enzyme digestion
  Organic extraction and enzyme digestion

 Protease such as pronase or proteinase K before phenol extraction is

required for samples rich in proteins or if phenol: chloroform extraction is
not sufficient

 Proteases break polypeptides into smaller units, which are more easily
removed by phenol

 Some RNA molecules can be effectively removed by the enzyme

ribonuclease, which rapidly degrades these molecules into ribonucleotide
subunits
 Non-organic DNA extraction

• Does not use organic reagents such as phenol or

chloroform
• Digested proteins are removed by salting out with high
concentrations of LiCl
• If salts are not adequately removed, problems could
occur with the molecular marker analysis e.g. RFLP
 Non-organic DNA extraction procedure

1. Cell Lysis Buffer - lyse cell membrane, nuclei are intact,

pellet nuclei
2. Resuspend nuclei in Protein Lysis Buffer containing a high
concentration of Proteinase K. Lyse nuclear membrane
and digest protein at 65C for 2 hours. Temperature helps
denature proteins, and Proteinase K auto digests itself
3. To remove proteinaceous material, LiCl is added to a final
concentration of 2.5 M, and incubated on ice. Proteins
precipitate out and are pelleted by centrifugation
 Non-organic DNA extraction procedure

4. DNA remains in solution and supernatant is

transferred to a new tube, care must be taken not to
take any of protein pellet
5. DNA is precipitated
6. Precipitated DNA is washed with 70% ethanol, dried
under vacuum and re-suspended in Tris-EDTA (TE)
buffer and stored.
 Concentrating DNA samples
 The most frequently used method of concentration is ethanol
precipitation
 In the presence of salt and at a temperature of −20°C or less, absolute
ethanol efficiently precipitates polymeric nucleic acids
• With a thick solution of DNA the ethanol can be layered on top of the
sample causing molecules to precipitate at the interface
• A glass rod is pushed through the ethanol into the DNA solution.
When the rod is removed DNA molecules adhere and can be pulled
out of the solution in the form of a long fiber
 Alternatively, if ethanol is mixed with a dilute DNA solution, the
precipitate can be collected by centrifugation, and then re-dissolved in
an appropriate volume of water (nuclease free) or buffer
Concentrating of DNA samples
 Differential extraction of DNA:
separation of male and female DNA
 Differential Extraction
• Process to isolate the male and female DNA from a sexual assault
evidentiary sample
• From a single evidentiary sample, a female fraction containing the
DNA from the victims epithelial cells, and a male fraction containing
the DNA from the sperm of the assailant are isolated
• Preferential breaking open the female epithelial cells with an
incubation in a SDS/Proteinase K mixture
• Sperm heads remain intact during this incubation
 Differential Extraction
• The sperm heads are pelleted and the supernatant containing the female
fraction is collected and saved
• The sperm pellet is washed several times to remove any residual DNA
from the victim
• The sperm are subsequently lysed by treatment with a SDS/proteinase K/
dithiothreitol (DTT) mixture
• The DTT is required to breakdown (reduce) the protein disulfide bridges
that make up the sperm head
• The sperm are impervious to lysis without the addition of the DTT
• Both the male fraction and the female fraction are then extracted with
phenol-chloroform, and the DNA precipitated with ethanol
Differential Extraction
 Preparation of plasmid DNA

• A culture of cells, containing plasmids>> grown in liquid

medium>> harvested >> cell extract prepared
• The protein and RNA are removed >> the DNA
concentrated by ethanol precipitation
• Separation of the plasmid DNA from the large amount of
bacterial chromosomal DNA is essential
• The methods for separation of plasmids are based on the
several physical differences between plasmid DNA and
bacterial DNA
 Preparation of plasmid DNA

Important considerations during plasmid DNA purification:

• Size: Plasmids are very small compared to

chromosomal DNA. Techniques for small plasmid DNA
separation should be sensitive and effectively purify
plasmid DNA

• Conformation: Circular vs. linear DNA

 Separation of Plasmid DNA on the basis of size

• If the cells are lysed under very carefully controlled conditions, only a minimal
amount of chromosomal DNA breakage occurs

• The resulting DNA fragments are still very large—much larger than the plasmids—
and can be removed with the cell debris by centrifugation

• Bacterial chromosome is physically attached to the cell envelope, so fragments of

the chromosome sediment with the cell debris if these attachments are not broken
 Separation of Plasmid DNA on the basis of size

• Cell disruption must be carried out very gently to prevent wholesale breakage of the
bacterial genomic DNA
• Treatment with EDTA and lysozyme is carried out in the presence of sucrose (prevents
the cells from bursting immediately)
• Sphaeroplasts are formed, cells with partially degraded cell walls that retain an intact
cytoplasmic membrane
• Cell lysis is then induced by adding a non-ionic detergent such as Triton X-100 (causes
very little breakage of the bacterial DNA which is precipitated with cell wall)
• Centrifugation leaves a cleared lysate, consisting almost entirely of plasmid DNA
Separation of Plasmid DNA
on the basis of size
 Separation of Palsmid DNA
on the basis of conformation

• Most plasmids exist in the cell as supercoiled

molecules

• The supercoiled conformation can be maintained

only if both polynucleotide strands are intact, hence
the more technical name of covalently closed
circular (ccc) DNA

• If one of the polynucleotide strands is broken the

double helix reverts to its normal relaxed state, and
the plasmid takes on the alternative conformation,
called open-circular (oc)
 Separation of Palsmid DNA
on the basis of conformation

 Two methods are most widely used to

separate this supercoiled plasmid DNA-

a) Alkaline denaturation

b) Ethidium bromide–caesium chloride density

gradient centrifugation
 Alkaline Denaturation
• At a narrow pH range at which non-supercoiled DNA is
denatured whereas supercoiled plasmids are not

• Sodium hydroxide is added to a cell extract or cleared lysate

(adjust pH to 12.0–12.5) >> the hydrogen bonding in non-
supercoiled DNA molecules is broken, causing the double
helix to unwind and the two polynucleotide chains to
separate

• Next, acid is added >> the denatured bacterial DNA strands

re-aggregate into a tangled mass which is pelleted by
centrifugation, leaving plasmid DNA in the supernatant
Alkaline Denaturation
 Advantages of Alkaline Denaturation

• Most of the protein and RNA becomes insoluble and

can be removed by centrifugation steps (If the cell is
lysed using SDS and then neutralized by sodium
acetate)

• Phenol extration and ribonuclease treatment is not

needed in alkaline denaturation method
 Ethidium bromide–caesium chloride density gradient centrifugation

• A specialized version of the more general technique of equilibrium or density

gradient centrifugation, can separate DNA, RNA, and protein and is an alternative to
organic extraction or column chromatography for DNA purification

• A density gradient is produced by centrifuging a solution of caesium chloride (CsCl)

at a very high speed

• Macromolecules present in the CsCl solution when it is centrifuged form bands at

distinct points in the gradient

• Exactly where a particular molecule bands depends on its buoyant density

Ethidium bromide–caesium chloride density gradient centrifugation

• DNA has a buoyant density of about 1.70 g/cm3, and

therefore migrates to the point in the gradient where the
CsCl density is also 1.70 g/cm3

• In contrast, protein molecules have much lower buoyant

densities, and so float at the top of the tube, RNA forms
a pellet at the bottom
Ethidium bromide–caesium chloride density gradient centrifugation
 Ethidium bromide–caesium chloride
density gradient centrifugation

• Density gradient centrifugation in the presence of Ethidium

Bromide (EtBr) can be used to separate supercoiled DNA from
non-supercoiled molecules

• Ethidium bromide binds to DNA molecules by intercalating

between adjacent base pairs, causing partial unwinding of the
double helix

• The unwinding result in a decrease in the buoyant density, by

as much as 0.125 g/cm3 for linear DNA
 Ethidium bromide–caesium chloride
density gradient centrifugation

• Supercoiled DNA, with no free ends, has very little freedom to unwind and can only
bind a limited amount of EtBr

• The decrease in buoyant density of a supercoiled molecule is therefore much less,

only about 0.085 g/cm3

• The supercoiled molecules form a band in an EtBr–CsCl gradient at a different

position to linear and open-circular DNA
 Ethidium bromide–caesium chloride
density gradient centrifugation
• When a cleared lysate is subjected to this procedure, plasmids
band at a distinct point, separated from the linear bacterial DNA,
with the protein floating on the top of the gradient and RNA
pelleted at the bottom

• The position of the DNA bands can be seen by shining ultraviolet

radiation on the tube, which causes the bound EtBr to fluoresce

• The pure plasmid DNA is removed by puncturing the side of the

tube and withdrawing a sample with a syringe

• The EtBr bound to the plasmid DNA is extracted with n-butanol

and the CsCl removed by dialysis
Ethidium bromide–caesium chloride
density gradient centrifugation
  Preparation of plasmid DNA can be hindered
by the fact that plasmids make up only a
small proportion of the total DNA in the
bacterial cell

 The yield of DNA from a bacterial culture

may therefore be disappointingly low

Plasmid amplification offers a means of increasing this yield

 Plasmid amplification
• The aim of amplification is to increase the copy number of a
plasmid
• Some multicopy plasmids (those with copy numbers of 20 or more)
have the useful property of being able to replicate in the absence of
protein synthesis
• This property can be utilized during the growth of a bacterial
culture for plasmid DNA purification
• After a satisfactory cell density has been reached, an inhibitor of
protein synthesis (e.g., chloramphenicol) is added, and the culture
incubated for a further 12 hours
• During this time the plasmid molecules continue to replicate, even
though chromosome replication and cell division are blocked
• In this way the several thousand copies of plasmids can be
produced for genetic engineering
Plasmid amplification
 Preparation of bacteriophage DNA

Life cycle of
Bacteriophage
lambda
 Preparation of bacteriophage DNA

• The key difference between phage DNA purification and the

preparation of either total cell DNA or plasmid DNA is that for
phages the starting material is not normally a cell extract
• Bacteriophage particles can be obtained in large numbers
from the extracellular medium of an infected bacterial culture
• When a bacterial culture is centrifuged, the bacteria are
pelleted, leaving the phage particles in suspension
• The phage particles are then collected from the suspension
and their DNA extracted by a single deproteinization step to
remove the phage capsid
Preparation of bacteriophage DNA
 Preparation of bacteriophage DNA

Purification of phage DNA is very difficult. Because-

a) Growing an infected bacterial culture in such a way that the

extracellular phage titer (the number of phage particles per
ml of culture) is sufficiently high
-In practical terms, the maximum titer that can
reasonably be expected for lambda is 1010 per ml which
will yield only 500 ng of DNA

b) Large culture volumes, in the range of 500–1000 ml, are

needed if substantial quantities of phage DNA are to be
obtained
 Growth of cultures to obtain a high ʎ titer

• The naturally occurring ʎ phage is lysogenic, and an infected culture consists mainly
of cells carrying the prophage integrated into the bacterial DNA

• To get a high yield of extracellular λ, the culture must be induced, so that all the
bacteria enter the lytic phase of the infection cycle, resulting in cell death and
release of λ particles into the medium

• Most laboratory strains of λ carry a temperature-sensitive (ts) mutation in the cI

(reressor) gene for maintaining the phage in the integrated state
 Growth of cultures to obtain a high ʎ titer
• If inactivated by a mutation, the cI gene no longer functions
correctly and the switch to lysis occurs
• In the cIts mutation, the cI gene is functional at 30°C, at
which temperature normal lysogeny can occur
• But at 42°C, the cIts gene product does not work properly, and
lysogeny cannot be maintained
• A culture of E. coli infected with a λ phages carrying the cIts
mutation can therefore be induced to produce extracellular
phages by transferring from 30°C to 42°C
Growth of cultures to obtain a high ʎ titer
 Preparation of non-lysogenic lambda phages

• Although most λ strains are lysogenic, many cloning vectors derived from λ are
modified, by deletions of the cI and other genes, so that lysogeny never occurs

• These phages cannot integrate into the bacterial genome and can infect cells only by
a lytic cycle

• The key to obtaining a high titer lies in the way in which the culture is grown, in
particular the stage at which the cells are infected by adding phage particles
 Preparation of non-lysogenic lambda phages

• If phages are added before the cells are dividing at their

maximal rate, then all the cells are lysed very quickly, resulting
in a low titer

• If the cell density is too high when the phages are added, then
the culture will never be completely lysed, and again the
phage titer will be low

• The ideal situation is when the age of the culture, and the size
of the phage inoculum, are balanced such that the culture
continues to grow, but eventually all the cells are infected and
lysed
Preparation of non-lysogenic λ phages
 Collection of phages from an infected culture
• The remains of lysed bacterial cells, along with any intact cells
that are inadvertently left over, can be removed from an infected
culture by centrifugation, leaving the phage particles in
suspension

• Collection of phages is usually achieved by precipitation with

polyethylene glycol (PEG)

• PEG is a long chain polymeric compound, that in the presence of

salt, absorbs water and causes macromolecular assemblies such
as phage particles to precipitate

• The precipitate can then be collected by centrifugation

Collection of phages from an infected culture
 Purification of DNA from ʎ phage particles
• The PEG precipitate also contains a certain amount of
bacterial debris, possibly including unwanted cellular DNA

• The contaminants can be separated from the ʎ particles by

CsCl density gradient centrifugation

• The λ particles band in a CsCl gradient at 1.45–1.50 g/cm3 and

can be withdrawn from the gradient (as described previously
for DNA bands)

• Removal of CsCl by dialysis leaves a pure phage preparation

from which the DNA can be extracted by either phenol or
protease treatment to digest the phage protein coat
Purification of DNA from ʎ phage particles
Purification of M13 DNA, a filamentous phage
 Purification of M13 DNA

Advantages of M13 over λ DNA preparation

• The double-stranded replicative form of M13, behaves like a high copy

number plasmid

• Very easily purified by the standard procedures for plasmid preparation

• The infected cells continually secrete M13 particles into the medium, with lysis
never occurring, a high M13 titer is achieved simply by growing the infected
culture to a high cell density

• No problem with cell debris contaminating the phage suspension (the CsCl
density gradient centrifugation step, needed for lambda phage preparation, is
rarely required with M13)
 Purification of M13 DNA

• A cell extract is prepared from cells infected with M13, and the replicative form
separated from bacterial DNA by, EtBr–CsCl density gradient centrifugation

• The single-stranded form of the M13 genome, are very easy to obtain (very useful
for sequencing)
 Purification of M13 DNA
• Single-stranded M13 DNA preparation involves growth of a small volume of infected
culture, centrifugation to pellet the bacteria, precipitation of the phage particles
with PEG, phenol extraction to remove the phage protein coats, and ethanol
precipitation to concentrate the resulting DNA

• A cell titters of 1012 / ml and above are quite easy to obtain and thus, a significant
amounts of single-stranded M13 DNA can be prepared from cultures of small
volume—5 ml or less
Purification of M13 DNA causes few problems
 Purification of RNA

• Requires STRICT precautions to avoid sample degradation

• RNA especially labile
• RNases are naturally occurring enzymes that degrade RNA
• Common laboratory contaminants (from bacterial and
human sources) are common sources of RNAse
• RNase is also released from cellular compartments during
isolation of RNA from biological samples
• Can be difficult to inactivate
 RNA extraction: organic method

1. Lyse/homogenize cells
2. Add phenol:chloroform:isoamyl alcohol to lysed sample, and centrifuge
3. Organic phase separates from aqueous phase
– Organic solvents on bottom
– Aqueous phase on top (contains total RNA)
– Cellular debris and genomic DNA appears as a “film” of debris at the interface of
the two solutions
4. Remove RNA solution to a clean tube; precipitate RNA and wash with ethanol, then
resuspend RNA in water
 Affinity purification of RNA

1. Lyse cells, and spin to remove large particulates/cell debris

2. Apply lysate (containing nucleic acids and cellular
contaminants) to column with glass membrane
3. Wash with alcohol to remove contaminants; nucleic acids
stick to glass membrane/ column while contaminants wash
through
4. Treat with DNase enzyme to remove contaminating DNA
5. Apply water** to the column; purified RNA washes off the
glass and is collected
 Isolation of PolyA (messenger) RNA

• mRNA comprise only 2.5-5% of the total RNA pool

• mRNA molecules have a tail of A’s at the 3’ end (polyA tail)
• Oligo (dT) probes can be used to purify mRNA from other
RNAs
• mRNA can be eluted from oligo (dT) matrix using water or
low-salt buffer
 Measurement of DNA concentration
 DNA concentrations can be accurately measured by ultraviolet
(UV) absorbance spectrophotometry
 The amount of UV radiation absorbed by a solution of DNA is
directly proportional to the amount of DNA in the sample
 Usually absorbance is measured at 260 nm, at which
wavelength an absorbance of 1.0 corresponds to 50 mg of
double-stranded DNA per ml
 Ultraviolet absorbance can be used to check the purity of a DNA
preparation
 With a pure sample of DNA, the ratio of the absorbance at 260
and 280 nm (A260/A280) is 1.8.
 Ratios of less than (or grater than) 1.8 indicate that the
preparation is contaminated, either with protein or with phenol
 Detection and quantitation of nucleic acids

 Ethidium bromide

 Most widely used dye to quantitate DNA and RNA

 Intercalating agent
 Florescent intensity measured when exposed to UV
 Carcinogenic
 Detection and quantitation of nucleic acids

• DNA/RNA concentration can be measured at 260 nm in spectrophotometer

• OD 1= 50µg/mL of double stranded DNA (40 µg/mL for RNA)

• Convenient but not sensitive (protein / phenol interference)

• No check on the integrity of purified nucleic acids

 Gel electrophoresis

• Used for analysis and purification of DNA

• Separation of DNA based on size
• Concentration of agarose: at reduced concentration effective separation of
large fragments and at increased concentration effective separation of small
nucleic acid fragments
• Poly acrylamide gels used for separation of smaller molecules (only a few tens
of base pairs)
 Analytical gel electrophoresis
• Agarose gel electrophoresis can be used to analyse the
composition and quality of nucleic acids e.g. size of restriction
digest or PCR products

• A linear relationship exist between the logarithm size and

distance moved (by nucleic acids) in the gel

• Calibration of the gel is done by a marker (DNA ladder) of

known size and size of sample can be determined by
comparing with the ladder
Analytical gel electrophoresis
 Pulse field gel electrophoresis

• Agarose gel electrophoresis does not have enough resolving power to separate larger
DNA molecules

• Pulse field gel electrophoresis separates very large DNA molecules

• Polarity of the electric field is periodically reversed

• Each time the field is reversed the molecule is reoriented in order to move thorough the
pores in opposite directions

• Larger molecules have slower mobility

Pulse field gel electrophoresis
 Pulse field gel electrophoresis
• Successful application of PFGE (also called orthogonal
field alternation gel electrophoresis (OFAGE)) requires
the isolation of intact genomic DNA or extremely large
fragments

• It is extremely difficult to obtain intact large DNA

molecule during extraction

How to prepare large DNA (intact) for pulse field gel

electrophoresis?
 Cells are embedded in agarose gel (agarose plug) and cell
lysis and restriction digestions are carried out
 Agarose plug is inserted into gel and DNA fragments
move from plug to gel when electricity applied
 Electrophoresis for non-linear DNA
• Pasmids are circular but a nicked plasmid has a open
circular form and double stranded break causes a linear
form
• All three forms are of same size, mobility in a gel varies
 Electrophoresis for non-linear DNA

 Open circular form will move slowly than circular

or linear form of plasmids

 Mobility of the circular or linear form of plasmids

is difficult to predict: dependent on the size and
electrophoretic conditions

 For a purified plasmid preparation it is usual to

have 2/3 bands
 Preparative gel electrophoresis

• Used to purify a specific nucleic acid fragment from a complex mixture

• Low-melting point agarose used to recover DNA fragments
• After staining with ethidium bromide and exposed to UV
• Specific bands are cleaved off from the gel by a razor blade followed by
purification of the recovered fragment
 Electrophoresis for RNA
 For separation of RNA molecures denaturating gels (containing urea or
formaldehyde) are used
Thanks !!