Manipulation of

Nucleic Acids

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 Cutting and Rejoining DNA
• The chromosomes that carry our genes are extremely long
molecules of DNA.

• In order to manipulate DNA conveniently, it needs to be cut

into manageable lengths.

• The discovery of restriction enzymes, which occur naturally

in bacteria, allowed this.

• Restriction enzymes recognize specific sequences within a

DNA molecule and then make a double-stranded cut.

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• Restriction enzymes have been used for analyzing DNA
structure and for molecular cloning.

• Indeed, the discovery and use of restriction enzymes

was largely responsible for the emergence of genetic
engineering.

• Nowadays, the use of restriction enzymes has often

been displaced by more sophisticated techniques such
as DNA sequencing or the polymerase chain reaction
(PCR).

• Nonetheless, restriction enzymes are still used in a

variety of situations.

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 Restriction and Modification of DNA

• Nucleases are enzymes that degrade nucleic acids.

• They are categorized as ribonucleases (RNases) that attack

RNA and deoxyribonucleases (DNases) that attack DNA.

• Most nucleases are specific, though the degree of

specificity varies greatly.

• Some nucleases will only attack single-stranded nucleic

acids, others will only attack double-stranded nucleic acids,
and a few will attack either kind.

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• Exonucleases attack the ends of nucleic acid molecules
and usually remove just a single nucleotide.

• Exonucleases can attack either the 3’-end or the 5’-end

but not both.

• Endonucleases cleave the nucleic acid chain in the

middle.

• Some endonucleases are nonspecific; others, in

particular the restriction enzymes, are extremely
specific and will only cut DNA after binding to specific
recognition sequences.

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• In nature, foreign DNA entering a bacterial cell is most likely due to
virus infection, and natural defense systems have evolved to
combat the infection.

• When viruses attack bacteria, the virus protein coat is left outside
and only the virus DNA enters the target cell.

• Bacteria produce restriction enzymes (sometimes called restriction

endonucleases) to destroy foreign DNA.

• These are endonucleases that recognize specific sequences of four

to eight nucleotides in length known as recognition sites.

• The restriction enzymes cut both phosphate backbones thus cutting

the target DNA molecule in two.

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• Restriction enzymes must distinguish bacterial DNA from invading
DNA because the key to successful defense is to degrade the
foreign DNA without endangering the cell’s own DNA.

• To protect cellular DNA, modification enzymes bind to the same

recognition sites as the corresponding restriction enzymes and add
a methyl group to the DNA.

• Modification enzymes usually add the methyl group to adenine or

cytosine within the recognition site, and this modification prevents
the corresponding restriction enzyme from recognizing the site.

• Consequently, the bacterial DNA, either chromosomal or plasmid, is

immune to that cell’s own restriction enzymes.

• In contrast, incoming, unmodified DNA will be degraded.

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 Recognition of DNA by Restriction Endonucleases

• Due to their ability to recognize specific sites in

DNA, restriction endonucleases have become one
of the most widely used tools in genetic
engineering.

• Restriction enzyme recognition sites are usually

four, six, or eight bases long and the sequence
forms an inverted repeat.

• Thus, the sequence on the top strand of the DNA

is the same as the sequence of the bottom strand
read in the reverse direction.
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• Several hundred different restriction enzymes are now known and
each has its own specific recognition site.

• Some recognition sites require a specific base at each position.

Others are less specific and may require only a purine or a
pyrimidine at a particular position.

• Since any random sequence of four bases will be found quite

frequently, four base-recognizing enzymes cut DNA into many short
fragments.

• Conversely, since any particular eight-base sequence is less likely,

the eight-base-recognizing enzymes cut DNA only at longer
intervals and generate fewer larger pieces.

• The six-base enzymes are the most convenient in practice, as they

give an intermediate result.

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 Naming of Restriction Enzymes

• Restriction enzymes have names derived from the initials of the bacteria
they come from.

• The first letter of the genus name is capitalized and followed by the first
two letters of the species name (consequently, these three letters are in
italics).

• The strain is sometimes represented (e.g., the R in EcoRI refers to E. coli

strain RY13).

• The roman letter indicates the number of restriction enzymes found in the
same species.

• For example, Moraxella bovis has two different restriction enzymes called
MboI and MboII.

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 Cutting of DNA by Restriction Enzymes

• It might seem logical for the DNA to be cut at the

recognition site where the restriction enzyme
binds.

• This is often true, but not always.

• There are two major types of restriction enzyme

that differ in where they cut the DNA, relative to
the recognition site.

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• Type I restriction enzymes cut the DNA a
thousand or more base pairs away from the
recognition site.

• Since the exact length of the loop is not

constant, and since the base sequence at the
cut site is not fixed, these enzymes are of little
practical use to molecular biologists.

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• Type II restriction enzymes cut the DNA in the middle of the
recognition site.

• Since the exact position of the cut is known, these restriction

enzymes are typically used in genetic engineering.

• There are two different ways of cutting the recognition site in half.

• One way is to cut both strands of the double-stranded DNA at the

same point. This leaves blunt ends.

• The alternative is to cut the two strands in different places, which

generates overhanging ends.

• The ends made by such a staggered cut will base pair with each
other and are known as sticky ends.

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• If two different pieces of DNA are cut with the same
restriction enzyme or with different enzymes that
generate the same overhang, the same sticky ends are
generated.

• This allows fragments of DNA from two different

original DNA molecules to be bound together by
matching the sticky ends.

• Such pairing is temporary since the pieces of DNA are

only held together by hydrogen bonding between the
base pairs, not by permanent covalent bonds.

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• Nonetheless, this assists the permanent
linking of the sugar-phosphate backbone by
DNA ligase.

• When two sticky ends made by the same

enzyme are ligated, the junction may be cut
apart later by using the same enzyme again.

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 DNA Fragments Are Joined by DNA Ligase

• To link segments of DNA together

permanently, the enzyme DNA ligase is used.

• If DNA ligase finds two DNA fragments

touching each other end to end, it will ligate
them together.

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 Making a Restriction Map

• A diagram that shows the location of restriction enzyme cut sites on

a segment of DNA is known as a restriction map.

• Today, such maps would probably be made by sequencing the DNA

and scanning the sequence for recognition sites.

• In the early days of molecular biology, restriction maps were

derived experimentally.

• The DNA was digested with a series of restriction enzymes, one at a

time.

• The fragments of digested DNA were separated by agarose gel

electrophoresis.

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• This reveals how many recognition sites each
enzyme has in the DNA and their distances
apart.

• This approach allows the construction of a

complete restriction map of any segment of
DNA.

• This may then be used as a guide for further

manipulations

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 Restriction Fragment Length Polymorphisms (RFLPs)

• Related molecules of DNA, such as different

versions of the same gene from two related
organisms, normally have very similar sequences.

• Consequently, they will have similar restriction

maps.

• However, occasional differences in base sequence

will result in corresponding differences in
restriction sites.
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• Each restriction enzyme recognizes a specific
sequence (usually of four, six, or eight bases).

• If even a single base within this recognition

sequence is altered, the enzyme will no longer
cut the DNA.

• Consequently, restriction sites that are

present in one version of a sequence may be
missing in its close relatives.

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• If two such related but different DNA molecules are cut with the
same restriction enzyme, segments of different lengths are
produced.

• When these are separated on a gel, bands of different sizes will

appear.

• A difference between two DNA sequences that affects a restriction

site is known as a restriction fragment length polymorphism (RFLP).

• RFLPs may be used to identify organisms or analyze relationships

even if we do not know the function of the altered segment of
DNA.

• RFLPs were once widely used in forensic analysis but have now
been largely replaced by methods based on PCR.

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 Measuring DNA and RNA Concentration With
Ultraviolet Light
• The aromatic rings of the bases found in DNA and RNA absorb ultraviolet
(UV) light with an absorption maximum at 260 nm.

• If a beam of UV light is shone through a solution containing nucleic acids,

the proportion of the UV absorbed depends on the amount of DNA or
RNA.

• This approach is widely used to measure the concentration of a solution of

DNA or RNA.

• The amount of UV light absorbed by a series of standard DNA

concentrations is measured to calibrate the technique.

• The amount of UV light absorbed by the unknown DNA is then determined

and plotted on the standard curve to deduce the concentration.

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 Radioactive Labeling of Nucleic Acids

• DNA from different sources can be distinguished by adding

a specific label to one sample.

• For example, when a virus attacks bacteria, the viral DNA

enters the bacteria.

• A scientist studying this process can distinguish the viral

DNA from the bacterial DNA by labeling one of the two.

• Originally, incorporating radioisotopes into the DNA of the

virus or bacteria was used to distinguish the two.

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• For example, radioactive nucleotides would be
provided to the bacteria so that during the next round
of DNA synthesis, some of the radioactive molecules
would be incorporated into the bacterial chromosome.

• The bacterial DNA would be “hot,” or radioactive, and

the viral DNA would be “cold,” or nonradioactive.

• Radioisotopes are the radioactive forms of an element.

• In molecular biology, two are especially important: The

radioactive isotopes of phosphorus, P32 , and sulfur, S35.

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• Nucleic acids consist of nucleotides linked
together by phosphate groups, each containing a
central phosphorus atom.

• If 32P is inserted at this position we have

radioactive DNA or RNA.

• The half life of 32P is 14 days, which means that

half of the radioactive phosphorus atoms will
have disintegrated during this time period, so
experiments using 32P need to be done fast!

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 Detection of Radioactively Labeled DNA

• The two methods most widely used in molecular

biology to measure radioactivity are scintillation
counting and autoradiography.

• If a sample is in liquid or on a strip of filter paper,

the amount of radioactivity is measured using
scintillation counting.

• If the sample is flat, such as an agarose gel,

scientists use autoradiography to pinpoint the
location of radioactive bands or spots.
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• Autoradiography is used for detecting
radioactively labeled DNA or RNA in a gel after
separation by electrophoresis.

• Gels containing RNA or DNA are transferred

onto membranes by blotting to allow more
convenient handling during autoradiography,
or the gel can be dried onto filter paper.

• Whether the radioactive DNA is in a dried gel,

on a membrane or piece of filter paper, the
radioactive isotope emits beta particles.
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• The particles will turn regular photographic film
black, exactly the same way that light turns
photographic film black.

• To see the location of radioactively labeled DNA,

a sheet of photographic film is laid on top of the
gel or filter and left for several hours or
sometimes even for days.

• The film darkens where the radioactive DNA

bands or spots are found.
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 Fluorescence in the Detection of DNA and RNA

• Most classic work in biochemistry and molecular

biology was done with radioactive tracers and probes.

• Although the low levels of radioactivity used in

laboratory analyses are little real hazard, the burden of
storage and proper disposal of the waste has made it
relatively cheaper and quicker to use other detection
methods.

• Some of the newer DNA detection methods use

fluorescent materials.
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• Fluorescence occurs when a molecule absorbs light of
one wavelength and emits light of lower energy at a
longer wavelength.

• Detection of fluorescence requires both a beam of light

to excite the dye and a photo-detector to detect the
fluorescent emission.

• A variety of fluorescent dyes are available to attach to

DNA, and each one has slightly different absorption
and emission wavelengths.

• The detectors can readily discern and record the

different tags making it possible to have multiple
fluorescent tags in one single sample.
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• The fluorescence activated cell sorter or FACS
was originally designed for sorting cells
labeled with a fluorescent tag from those that
were untagged.

• The new generation of more sensitive FACS

machines is capable of sorting chromosomes
labeled by hybridization with fluorescent
tagged DNA probes.

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 Chemical Tagging With Biotin or Digoxigenin

• Biotin (a vitamin) and digoxigenin (a steroid

from foxglove plants) are two molecular tags
widely used for labeling DNA.

• Both biotin and digoxigenin are linked to

uracil, which is normally a component of RNA.

• In order to label DNA with biotin or

digoxigenin, uracil must be incorporated into
DNA.
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• To achieve this, uridine triphosphate (UTP) is
modified to deoxyuridine triphosphate (deoxy
UTP).

• If deoxyUTP labeled with biotin or digoxigenin is

added to a DNA synthesis reaction, DNA
polymerase will incorporate the labeled uridine
where thymidine is normally inserted.

• The biotin or digoxigenin tags stick out from the

DNA without disrupting its structure.

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• Biotin and digoxigenin are not colored or fluorescent molecules, but
they can be detected in a two-stage process.

• The first step is to use a molecule that specifically recognizes biotin

or digoxigenin.

• Biotin is a vitamin required both by animals and many bacteria.

Chickens lay highly nutritious eggs that would be a paradise for
invading bacteria. One of the defense mechanisms to protect the
egg from bacterial attack is a protein known as avidin.

• This protein, found in egg white, binds biotin so avidly that invading
bacteria become vitamin deficient. Molecular biologists use avidin
to bind the biotin tag.

• Digoxigenin also requires a second detectable molecule. In this

case, an antibody that recognizes and binds digoxigenin is used.

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• The antibody or avidin molecules are easily visualized by a variety of
different methods.

• The first option is to attach an enzyme that generates a colored

product to the avidin or the antibody.

• For example, avidin can be conjugated to alkaline phosphatase, an

enzyme that snips phosphate groups from a wide range of
molecules.

• One substrate for alkaline phosphatase, an artificial chromogenic

substrate known as “X-phos,” produces a blue dye when cleaved.

• X-phos consists of a dye precursor bound to a phosphate group, and

when alkaline phosphatase cleaves the phosphate off, the dye
precursor is converted to a blue dye by oxygen in the air.

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• Another option to detect biotin/avidin or digoxigenin/antibody is to use
chemiluminescence.

• In this method, an enzyme that produces light by a chemical reaction is

used to identify the labeled DNA.

• Alkaline phosphatase is still conjugated to the avidin or antibody, but a

different substrate, called “lumi-phos,” is added.

• Lumi-phos consists of a light-emitting group bound to the phosphate

group.

• When alkaline phosphatase removes the phosphate, the unstable

luminescent group emits light.

• Detecting and recording the light is accomplished by using photographic

film if the DNA is on a filter or in a gel, or by an instrument capable of
detecting light emissions if the DNA is in a solution.

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 Hybridization of DNA and RNA

• The complementary nature of DNA allows the

two strands to melt apart into single strands as
temperature increases.

• If the temperature is then slowly cooled back to

normal, the two strands re-anneal matching
every adenine to thymine and every guanine to
cytosine.

• Suppose that two closely related DNA molecules

are melted and then mixed.
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• If the mixture is cooled, re-annealing will occur.

• Although the sequences may not match perfectly,

if they are similar enough, some base pairing will
occur between complementary strands from
different original DNA double helixes.

• The result will be the formation of hybrid DNA

molecules.

• The formation of hybrid DNA molecules has a

wide variety of uses.

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• The relatedness of two DNA molecules can be assayed.

• In the figure, a sample of the first DNA molecule is

heated to melt it into single-stranded DNA.

• The single strands are then attached to a filter.

• Next, the filter is treated chemically to block any

remaining sites that would bind DNA.

• Then, a second single-stranded DNA sample is poured

on top of the filter.

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• If the second DNA has any related sequences, then a hybrid
will form on the membrane.

• The more closely related the two molecules are, the more
hybrid molecules will be formed and the higher the
proportion of the labeled DNA that will consequently be
bound to the filter.

• For example, if the DNA for a human gene, such as

hemoglobin, was fully melted and bound to a filter, then
DNA for the same gene but from different animals could be
tested.

• We might expect gorilla DNA to bind strongly, frog DNA to

bind weakly, and mouse DNA to be intermediate.

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• Another use for hybridization is in isolating genes for cloning.

• Suppose we already have the human hemoglobin gene and want to

isolate the corresponding gorilla gene.

• First, the human DNA is bound to the filter as before. Then gorilla
DNA is cut into short segments with a restriction enzyme.

• The gorilla DNA is heated to melt it into single strands and poured
over the filter.

• The DNA fragment that carries the gorilla gene for hemoglobin will
bind to the human hemoglobin gene and remain stuck to the filter.

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• A wide range of methods based on hybridization is used for
analysis in molecular biology.

• The basic idea in each case is that a known DNA sequence

acts as a “probe.”

• Generally, the probe molecule is labeled by radioactivity or

fluorescence for ease of detection.

• The probe is used to search for identical or similar

sequences in the experimental sample of target molecules.

• Both the probe and the target DNA must be treated to give
single-stranded DNA molecules that can hybridize to each
other by base pairing.

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 Southern, Northern, and Western Blotting

• Isolating new genes from related species provides a wealth

of information.

• Scientists that are studying human genes may need to find

similar genes in other organisms such as Drosophila,
zebrafish, or mice.

• Many scientists use Southern blotting to identify

corresponding genes in a different organism.

• Southern blotting is a technique in which one DNA sample

(target) is hybridized to another DNA sample (probe).

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• Suppose we have a large DNA molecule, such as a yeast
chromosome, and we wish to locate a gene whose
sequence is similar to a human gene of interest.

• First, the target or yeast DNA is cut with a restriction

enzyme, and the fragments are separated by gel
electrophoresis.

• The double-stranded fragments are melted into single-

stranded fragments by soaking the gel in alkaline
denaturing solution such as sodium hydroxide.

• Then the DNA fragments are transferred to a nylon

membrane.

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• Finally, the membrane is dipped in a solution of labeled DNA probe
molecules—in this example, a radioactively labeled segment of the
human gene.

• The probe binds only to those fragments with similar sequence.

• The filter will be “hot” or radioactive in the area where the probe
hybridizes to a matching sequence in the target DNA.

• A piece of photographic film is placed on the membrane and a black

spot will appear corresponding to the hybrid molecule.

• Note that Southern blotting only refers to hybridization of DNA to

DNA.

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• Although Southern blotting was named after its inventor, Edward
Southern, it set a geographical trend for naming other hybridization
techniques.

• Northern blotting refers to hybridization with RNA as the target molecule

and DNA as a probe.

• For example, DNA probes may be used to locate messenger RNA

molecules that correspond to the same gene.

• The mixture of RNA is run on a gel and transferred to a membrane.

• The membrane is then probed as earlier.

• Western blotting does not involve nucleic acid hybridization.

• Proteins are separated on a gel, transferred to a membrane, and detected

by antibodies.

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 Fluorescence in Situ Hybridization (FISH)

• All the previous techniques require the scientist

to isolate the DNA, RNA, or protein from its
cellular environment.

• In contrast, fluorescence in situ hybridization,

better known as FISH, detects the presence of a
gene, or corresponding messenger RNA, within
the cell itself.

• DNA sequences from the gene of interest must

first be generated for use as a probe. 65
• These may be obtained by cloning the gene or, more
usually nowadays, amplified by PCR.

• As the name indicates, the probe DNA is labeled with a

fluorescent dye whose localization will eventually be
observed under a fluorescence microscope.

• The tissue or cell must also be treated to denature the

target DNA, but this is done on the actual tissue, leaving
the DNA in its original location.

• If RNA is the target, the cells do not have to be treated with

denaturants since RNA is already single-stranded.

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• A thin section of tissue from a particular animal, a
mouse for example, may be treated with a DNA probe
for a known mouse gene.

• In this case, the probe will hybridize to the DNA in the

nucleus of all the cells.

• This tells us that the genes are in the nucleus, which

we knew anyway.

• More useful is using a DNA probe from one organism

to check for the presence of the same or similar genes
in related organisms, or in chimeric organisms created
by genetic engineering.

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• The probe does not need to represent coding
DNA and FISH can be used to search for any
known DNA sequences, including repetitive
DNA or transposable elements etc.

• For example, using virus DNA as a probe

reveals which cells contain virus genes, and
hence may be infected with virus, and
whether the virus genes are in the cytoplasm
or have penetrated the nucleus.

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• FISH can be used to identify where a particular gene is in
metaphase chromosomes.

• First, a chromosome smear is made on a microscope slide,

and then probed with a fluorescently labeled gene of
interest.

• The place where the probe binds reveals which

chromosome carries the gene corresponding to the probe.

• With sufficiently sophisticated equipment, the gene may be

localized to a specific region on the chromosome.

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