1

These next slides are background revision of gene and nucleic acid structure; and an
introduc7on to the major method of analysing isolated DNA and RNA, namely,
Agarose Gel Electrophoresis.

2
On the leE, the original X-ray image of a double-stranded DNA helix with the even
diameter depicted. The dark shading top and boIom represents the sugar
phosphates on the outside of the double helix, and the crosses in the centre are the
stacked H-bonded base pairs. Right panel shows the scien7sts with their wire-frame
model. The analogy of a winding staircase is oEen cited, with the bannister rails
represen7ng the sugar phosphate backbone and the steps as the base pairs in the
core of the molecule.

3
The five major bases in nucleic acids. The purines have a double ring with the larger
one being the same as the single ring in the pyrimidine bases. Thymine is only found
in DNA. Uracil is the Thymine equivalent in RNA. The “bases” are very weakly
alkaline, non-polar and absorb UV light at a maximum wavelength of 260 nm.

4
The 2 purine and 3 pyrimidine bases that give DNA and RNA their uniqueness (Uracil
is only found in RNA; Thymine in DNA). The contribu7ng sugars and phosphate are
also shown. Note that DNA has deoxyribose and RNA ribose. These sugars differ in
the 2-prime hydroxyl posi7on. The posi7ons are numbered according to the sugar
carbon posi7ons as indicated in the slide. Deoxyribose is really 2-deoxyribose, but is
usually abbreviated as ‘deoxyribose’. ‘Deoxy’ means ‘lacking oxygen’ and ribose is
converted to deoxyribose when the 2’-carbon of ribose loses its hydroxyl group (-
OH), leaving a hydrogen (H) in that posi7on.

5
Erwin Chargaff performed experiments in the 1930s that showed DNA to have equal
amounts of A and T; and of G and C. These equali7es became known as “Chargaff’s
Rules”. You can see from the diagram how the base pairings obey the rules. Note that
the A-T base pair (upper) has two H-bonds and the C-G pair three H-bonds. A third H-
bond between the H and CO in the upper pair is not formed due to the distance being
to great for the interac7on to occur. The limit of an H-bond is about 3.5 angstroms
(Å), which is approximately three and one half covalent bond distances. Note for
simplicity, this diagram does not show the phosphates that would be connected to
deoxyribose on the outside.

6
The leE panel diagram shows how nucleic acid (DNA or RNA) bases are paired by the
complementarity rule (Chargaff Rules) and this takes place across the double helix
and within its hydrophobic core. The sugar phosphate hydrophilic “backbone” of
each nucleo7de is posi7oned on the outside of each helix strand facing the aqueous
surroundings, making the whole molecule soluble and acidic. The small χαρτοον
(right panel) shows how the stacked base pairs gradually twist to give the nucleic acid
its helical shape. Each DNA or RNA nucleo7de consists of base, sugar, and phosphate.
Note the covalent aIachment of the nucleo7des in the ver7cal direc7on (on the
outside of the structure).

7
Base-pairing in nucleic acids. These pairings are always made, with each larger purine
base opposite a smaller pyrimidine one so that the DNA double-stranded molecule
has an even diameter. The base-pairing (dashed lines) occurs due to spontaneous
forma7on of H-bonds across the gap. Note the evenness of the diameter of the
paired bases that form the double helix. Note also the opposite polarity of the
strands, with each strand running in opposite direc7ons (leE strand 5’ to 3’; right
strand 3’ to 5’). This polarity is always the same and the double stranded nucleic acid
molecule will only form and remain stable in this opposite arrangement of the
strands. The 5’ designa7on refers to a free 5’-carbon on the first nucleo7de (5’-P at
the top of the leE strand). The 3’ designa7on refers to a free 3’-carbon on the last
nucleo7de of the strand (3’-OH at the boIom of the leE strand). Every nucleo7de is
joined covalently to the one above and below by phosphodiester bonds (see slide
18).

8
In these views of double-stranded DNA, note that there are H-bonds between the
nucleo7de bases in the centre of the molecule and it is predominantly these non-
covalent forces that hold the two strands together. The outside of the structure is
very polar due to the presence of the sugar (deoxyribose) and phosphates (nega7vely
charged) that surround the base pairs in the centre. DNA “dissolves” by solva7on
into water. Note also that the two strands are an7parallel. The two strands of DNA or
RNA are held together by the collec7ve ac7on of three types of non- covalent
interac7ng bonds: (i) H-bonds between the horizontally stacked base pairs in the core
of the molecule; (ii) hydrophobic interac7ons between the ver7cally stacked base
rings in the core; and (iii) sodium ions that neutralise the strong oxygen nega7ve
charges on the acidic phosphates on the outside of the molecules.

9
Two monophosphate nucleo7des joined with a 3’ to 5’ phosphodiester bond (water
is eliminated across the bond, just as it is in the pep:de bond between two amino
acids). The phosphodiester bond is formed by the elimina7on of the 3’-Carbon
hydroxyl group. These covalently joined nucleo7des are extended at either end with
new phosphodiester bonds between added nucleo7des, genera7ng a polynucleo7de
strand. If another an7parallel strand is added, then a double-stranded DNA or RNA
molecule is formed. Note that the 5-prime and 3-prime nomenclature derives from
the sugar (deoxyribose in DNA; ribose in RNA). Would the dinucleo7de above be
found in DNA or RNA?

10
A simple representa7on of base-pairing showing the precise (Chargaff Rules)
complementari7es of A with T and G with C in any DNA molecule. In RNA, it is A with
U (subs7tu7ng for ‘T’) and G with C. Note the an7parallel (5’ to 3’ and vice versa)
arrangement of the two strands.

11
We think of a gene in terms of the translated protein product, but all genes have
defined elements that control the expression of the gene (not shown here, but will
be illustrated in a later lecture), the ini7a7on of transcrip7on (by the binding of RNA
polymerase to the Promoter site), the ribosome binding sequence that directs the
RNA transcript to the protein transla7on site on the ribosome, and the termina7on
signal that enables RNA polymerase to be disengaged from the gene sequence. The
fourth element – the Open Reading Frame – is the set of triplet nucleo7de codons
that specify the sequence of amino acids for the translated polypep7de.

12
In eukaryotes, the genes in chromosomes are arranged differently to those in
prokaryotes. First and foremost are the controlling elements or regions upstream
(promoter) and downstream (termina7on machinery). The Open Reading Frame
(ORF) is non-con7guous (see upper diagram) and consists of a series of alternate
exons and introns. The exons are sec7ons of the ORF and comprise some of the
amino acid codons. The introns, also known as intervening sequences, are segments
of innocuous DNA that interrupt and act as bridges between successive exons. The
mRNA transcript made from these genes comprises the en7re exon-intron DNA gene
sequence. This pre-mRNA is then spliced within the nucleus to yield a mature mRNA
transcript (boIom of top diagram). This mRNA is now similar to those from
prokaryotes, in that it now comprises only the conjoined, uninterrupted exons with
the amino acid codons now con7guous in the manner of a normal ORF. Note, all
mRNAs (from whichever source) do not contain the promoter sequence, which is
back upstream (in the 5-prime direc7on) from the ORF. It is not transcribed because
its only purpose is in providing a landing spot for RNA polymerase to begin
transcribing the gene. The boIom diagram is an example of a collagen gene with
extensive exon-intron structure. We shall see in a later lecture how molecular cloning
gets around the problem of eukaryo7c gene structure and enables a copy of the gene
to be cloned and expressed in bacteria such as E. coli.

13
A DNA sequence begins and ends with non-coding nucleo7de bases (in black type).
An Open Reading Frame for a gene encodes a polypep7de, which is bookended by
Start and Stop codons (red), between which are the triplet codons (3 bases each,
shown here in alterna7ng colours) for the linear sequence (string) of amino acids that
will make up the polypep7de or protein for this gene.

One thing to note, but which is not shown here (will be dealt with in a later lecture),
is that any changes in the ORF will almost always be catastrophic in terms of the
expression of the protein. Dele7ng or inser7ng one or more bases will alter the ORF
and the protein will not be expressed (translated). One or two bases added or
substracted anywhere will ruin the reading frame (the correct types of amino acids in
the correct order). Insert or delete three bases and one amino acid will be removed
or added. While not always deleterious, this type of altera7on will almost always lead
to the protein not folding correctly or not working as it should.

14
This cartoon diagram contains an exaggerated representa7on of circular plasmid DNA
molecules that are only present in some bacteria and contain genes that give an
advantage to the host bacterium. The plasmid is replicated synchronously with the
chromosome and “inherited” by daughter cells along with the customary single
chromosome copy. Na7ve plasmids may occur in single to several copies per cell and
range in size from as small as 2 kilobases (kb) to perhaps over 400 kb. The E. coli
chromosome is 4000 kb in length and very large plasmids may reach almost 1/10th
the size of the chromosome. When we come to look at plasmids used in Molecular
Biology and Gene7c Engineering (Molecular Cloning; Recombinant DNA Technology),
we will see that only (hybrid) small plasmids are used and these have been
engineered to contain special features for molecular cloning.

15
Different views of ‘supercoils’.

16
This simple line drawing to scale shows the length of the bacterial E. coli
chromosome in rela7on to the size of the en7re E. coli cell. The chromosome can only
“fit” into the cell by being supercoiled so that it occupies a much smaller space. The
very thin diameter of the double-stranded DNA chromosome helps the supercoils to
be wound into a much smaller bundle (for a comparison, consider the size of a coIon
reel, which is about the size of a golf ball, with the length of the coIon spooled onto
the reel – about 120 metres!

17
DNA replica7on is a simple concept, but a complex process involving a
“choreographic performance” by a number of enzymes and enzyme complexes. In
this simple representa7on, the parental DNA strands (in green) are relaxed and
unwound to provide separate template strands for the replica7on process. In
bacterial the mul7subunit enzyme DNA Polymerase III catalyses the addi7on and
linking of nucleo7des into new polynucleo7de strands (in blue) that are
complementary to the template strands against which they are copied.

18
This is the standard method for iden7fying and analysing nucleic acids (DNA, RNA). It
is also known as submarine gel electrophoresis as the gel is submerged beneath the
running buffer. The nucleic acid samples are loaded in special dye mix that contains
marker dyes to indicate the extent of the run – these dyes run ahead of
(bromophenol blue, BPB) or with (xylene cyanol) nucleic acids of various sizes. The
loading dyes also contain nuclease inhibitors (EDTA) to prevent diges7on of DNA
during the run, and also contain a percentage of sucrose or a high MW polymer to
give the sample dye mix viscosity so that it will seIle and remain in the well un7l the
run is started. Gels are run un7l the BPB dye reaches at least halfway or near the end
of the gel. Note that the DNA is added at the cathode (nega7ve) end as the DNA has a
net nega7ve charge at the pH of the buffer (~7.5) and will therefore be conducted
towards the anode by the nega7vely charged buffer ions (and by their own –ve
charges). The DNA molecules are conducted through the regularly sized pores of the
gel. The migra7on rates of nucleic acid molecules is determined by their size and
conforma7on (see next slides).

19
The pore size (on the right) is determined by the agarose gel percentage. Most gels
are cast in the range of 0.5 to 3.0%. Higher gel percentages produce smaller pore
sizes and will only allow ready migra7on of small fragments. Lower percentage gels
will have larger pores and allow larger nucleic acids to migrate with rela7vely minimal
fric7onal resistance. In MB1 All gels will be around 0.7%. Agarose is a purified
component of agar that melts (100oC) and sets (~45oC) in the same way as agar.

20
Loading/running dye mixture is added to DNA or RNA samples in aqueous buffer.
Typically, about 5 µl dye to 15 µl sample. The loading dye mixture is supplied or made
at ~5x strength so only a few µls are required per sample. It contains buffer, EDTA,
bromophenol blue dye (and oEen a second dye xylene cyanol, which is green and
runs slower than bromophenol blue), and something to make the dye mix viscous,
usually 30% sucrose or a high MW polysaccharide. When the sample is added to the
well via a pipeIe 7p (as in the figure above), it drops into the well as it is more dense
than the running buffer; and it remains there un7l the current is applied. The drawing
at the boIom depicts the resolving of the DNA fragment bands in the gel as they
migrate from cathode (-) to anode (+) (middle panel). and the completed run with the
fragments arranged in maximum to minimum sizes from top to boIom. The gel
photo at the boIom of this slide depicts standards in the leE lane and decreasing
amounts of the same sample in lanes 1 to 6. The point to no7ce about these samples
is the way the broadness of the bands bias the actual size of the DNA smaple.
Although not a precise issue, the actual size of the sample band is best taken from
lane 6.

21
As we will see in a later slide and in Experiment 1 of the Prac7cal course, es7ma7ng
the sizes of DNA fragments typically involves running the unknown DNA against a set
of standards of known sizes. A good es7mate of the sizes of the fragments is taken
from a standard curve of the linear standards versus distance migrated from the
origin. In the simple diagram above, it is easy to make a good guess of the
approximate size of the three unknown fragments, by eye. If nothing else, this at
least will tell you if your more accurate values from the standard curve are correct (as
each band lies between two standards of known sizes). The hundreds or thousands of
double-stranded DNA molecules in each band are all of the same size and
configura7on, which is why they are running at the same posi7on in the gel.

22
Structures of the two DNA and RNA stains for agarose gels. Ethidium bromide (EtBr;
leE) was the original stain used un7l several years ago (and s7ll used in some places).
It has been superseded by Gel-Red (right), which has the same degree of binding and
staining sensi7vity as EtBr, but is far less toxic and is now widely used instead of EtBr.
You can see from the structures above that they are compara7vely similar, with Gel-
Red ionised with Iodine (I) versus bromine(Br) for Ethidium. Gel-Red is virtually a
duplicate of the EtBr mul7ple ring structure with a long alipha7c connec7ng chain
between the rings. Both dyes are planar (flat) and are able to intercalate (see next
slides) between the stacked base pairs of double-stranded DNA or RNA, or against
single-stranded RNA. The posi7ve charge on the nitrogen (N+) allows the dye to bind
to the nucleic acid’s opposite charged nega7ve phosphates.

23
A third type of DNA staining dye, which as you can see form the structure on the leE,
will bind to DNA more or less in the same way as Gel-Red or ethidium bromide (small
posi7ve charge; intercala7ng aroma7c rings). DNA stained with Sybr Green can be
visualised by UV excita7on in the same way as the other dyes, though the excita7on
spectrum is at a higher wavelength. The one property of Sybr Green that differs from
the other dyes is that it does not bind as well to single stranded DNA or RNA
(fluorescence is 11x weaker).

24
DNA “bands” in an agarose gel aEer electrophoresis and stained with Sybr Green or
Gel-Red (Irradiate at 340nm, visualize Sybr Green at 650nm, Gel-Red at 540nm). The
molecules of DNA (or RNA) in the original sample added to the origin wells migrate in
the electric current. They are sorted by the current into bands according to their
mass and conforma7on so that all molecules of the nucleic acid in any one band are
the same. The stained gel is viewed on an ultraviolet (UV) light box with the UV lamp
set to irradiate the nucleic acids at a wavelength of 302 nm, whereupon the nucleic
acids absorb the UV light, fluoresce, and re-emit it at 540 nm in the red-orange visible
region of the light spectrum. Photographic records of the gel made be made and
saved as jpg or 7f files.

25
A cartoon representa7on of the intercala:ng dye binding of EtBr, Gel-Red, or Sybr
Green in double-stranded DNA. Note that the DNA “swells” when the dye is bound,
with the en7re molecule becoming less dense. Dye binding is easier in linear or
relaxed circular molecules, more difficult in supercoiled DNA due to the 7ghtly wound
DNA strands, making dye access harder. The planar rings “stack” within the core of
the structure like extra “stairs” in between the base pairs and this stacking is aided by
the similarity of the hydrophobic planar rings of the dyes to those of the DNA or RNA
bases.

26
This table gives approximate nucleic acid separa7on and resolu7on ranges for various
percentages of agarose or polyacrylamide gels. Note that for smaller DNA molecules,
gels must be cast with polyacrylamide to ensure the pores of the gel are small
enough to resolve the popula7on of DNA molecules. Agarose gels may be cast up to
4% to resolve small DNA fragments, but these high % gels are briIle and produce too
much heat (by resistance) which could distort the DNA bands. Note the different
posi7onal profiles of the same set of linear standards in increasing percentage
agarose gels depicted in the gel photo on the right. This is best illustrated by looking
at the top few standards in the different % gels – see how they are spread out in the
0.7% gel, whose larger pores allow bigger DNA molecules to migrate through the
pores with less fric7onal resistance. The 1.5% gel shows the same high mass DNA
bands “compressed”, that is less spread out and much closer to the origin, because
they are too large to migrate freely and encounter fric7onal resistance from the
smaller pores. Note also that unlike circular nucleic acid molecules, linear DNA
molecules migrate end-on-end, but their lengths s7ll prevent them from fiwng easily
through small pores where their flexibility and length make them bump con7nuous
into the walls of the pores.

27
The top lane has a set of linear standards running according to their mass/length
sizes only (not conforma7on in this case, as they all have the same conforma7on and
will migrate according to the inverse log10 of their mass – see next slide). A DNA band
of unknown size, but of the same conforma:on as the set of standards is run in the
boIom lane. Its molecular mass can be es7mated from a standard curve (see next
slide). Note: unless otherwise stated, when reference is made to circular or linear
DNA, the molecules are deemed to be double-stranded by implica7on, as this is the
“normal” state for most such molecules. Single stranded DNA or RNA will almost
always be spelled out as such. There is also the conven7on of abbrevia7ng the type
of nucleic acid, for example, as ds DNA or ds RNA or ss RNA, where ‘ds’ and ‘ss’ are
double-stranded and single-stranded, respec7vely.

28
A standard curve drawn by plowng the distances migrated from the origin to the
DNA bands in an agarose gel (abscissa axis) versus the log10 of molecular masses
(ordinate axis). Alterna7vely, this plot could have been drawn on semi-log paper by
plowng the MW values directly on the log scale and the distances on the linear scale.
The size of the unknown DNA molecule (from the previous slide) is es7mated from
the standard curve by drawing a ver7cal line from its distance migrated (boIom axis)
up to the standard line and across to the ordinate axis from which its molecular mass
is interpolated. An important point about standard curves is that because the pore
sizes in the gel are the same as determined by the agarose concentra7on used, a
broad range of standards will migrate in two (or more) modes as shown above. That
is the larger DNA bands run a liIle slower than the smaller bands – due to the
resistance caused by the pore size on the larger molecules – and this is reflected in
the standard line with two slightly different gradients. Note that because this is
determined by the gel pore size and the spread of DNA sizes, the standard “curve”
line is drawn through every point and is NOT drawn as a line of best fit. Note, this
type of plot can be drawn on plain graph paper using the log10MW values on the y-
axis; or on semi=log graph paper, with the DNA MWs ploIed directly onto the log y-
axis. The result should be the same except that the log value of the unknown needs
ot be converted back to a linear (non-log) number.

29
A fundamental principle of plasmid DNA structure and conforma7ons in cells and in
molecular biology. Typically, and when present, plasmids in bacterial cells are in the
supercoiled configura7on – just as chromosomes are in all cells (bacteria to human).
This is the so-called res7ng state and is a “packaging” device that cells use to store
DNA molecules (plasmids and chromosomes). The nicked configura7on, also known
as relaxed or open circle, is the unwound version of the supercoiled plasmid. Nicks
come about through irradia7on or accidental breakages form 7me to 7me, but the
relaxed configura7on is also “forced” during normal cellular func7on, not of the
en7re plasmid (or genome), but locally so that transcrip7on of a gene or many genes
is made possible. It also comes about during the replica7on cycle of the DNA
molecule. Plasmids and chromosomes are interconverted within cells by a family of
enzymes known as gyrases or topoisomerases. The linear configura7on of the
plasmid shown here is ar7ficial, and not found in bacterial cells. It is generated by a
double nick across both strands, either by physical breakage or by enzyme ac7vity.

30
How the first supercoil is introduced into a relaxed, open circular plasmid. The
scissors in this diagram is replaced in cells by a gyrase or topoisomerase. There are
two types of these “winding” enzymes, type I and type II. One type introduces
supercoils, the other removes them. In this way, cells are able to fully supercoil or
relax DNA molecules (plasmids or chromosomes) on demand.

31
This cartoon slide indicates how different conforma7ons (spa7al shapes) of DNA
molecules can affect their migra7on rates. Note, these three DNA types (supercoiled
plasmid, relaxed plasmid, linear plasmid) are exactly the same molecular mass size,
but migrate differently due to shape/conforma7on differences alone and this is
caused by resistance to movement through the pores of the gel. The floppier the
molecules, the higher is the resistance to movement through the restric7ve pores
(shown as even-sized green circles in the gel cross-sec7on). Note the cartoon of the
supercoiled plasmid makes them look “linear”, but they are actually “squashed” into
that apparent shape by the supercoiling effect on their conforma7on. Agarose gel
electrophoresis that displays profiles of three plasmid conforma7ons and the
es7ma7on of the size of the linear version, as per the last three slides, is Experiment
1 in the laboratory arm of the subject.

32
This slide is a follow up of the previous one and emphasizes the point about
conforma7on versus migra7on rate. Three conforma7ons of the same DNA plasmid
are shown, but only two of these (relaxed, supercoiled) are seen in bacterial cell
lysate prepara7ons (covered in a later lecture). The linear version is provided
ar7ficially in this case to illustrate the three different mobili7es.

33
Two simple running and staining principles are illustrated in this slide. A general rule
is that – all DNA molecules of the same conforma7on migrate in agarose gels to
posi7ons that are inversely related to the log10 of their molecular masses. The
corollary of this principle (see previous two slides) is that molecules of the same
molecular mass but of different conforma7ons, migrate at different rates that are
determined by their rela7ve densi7es. The bands in this gel show the degree of
binding of dye (EtBr or Gel-Red). If all bands are of the same conforma7on and are,
for example, a set of linear standards derived from the same source DNA molecule by
being fragmented into standard sizes (as depicted on the labelled DNA molecule at
boIom), then there is the SAME number of molecules in each band. The upper bands
will appear broader than the lower bands because they are longer molecules and
occupy more space in the gel; and they are brighter because they bind more of the
dye. The line at the foot of the slide gives the fragment lengths, but also imagine that
there are thousands of the same molecules fragmented into A-F segments. Note the
anomaly in the profile above for the ‘C’ and ‘D’ fragments. These two fragments do
not run separately, but are virtually merged together, as they are nearly iden7cal in
size/length (see boIom scaled DNA molecule). This effect is easily detected in the gel
profile because C and D are running together and are about double the width they
would be as single bands. They also bind twice the amount of dye. So the otherwise
smooth transi7onal reduc7on in sizes from top to boIom (A…..F) is “disrupted” by
the CD doublet band. The amount of dye bound increases with the length/mass of

34
The first gel panel is a reminder of the principle seen in the previous slide, but this
7me without the doublet effect. The second panel shows the two major circular
plasmid bands obtained from bacterial cell lysates, namely, supercoiled and relaxed.
Plasmid DNA is present in cells in the supercoiled conforma7on (vegeta7ve or res7ng
state). When plasmid DNA is isolate from cell lysates, some of the supercoils become
“nicked” by the physical stresses of the isola7on procedure (shaking, vortexing,
pipewng etc) and this causes breaks in some of the strands - usually only one strand
in the doubled-stranded supercoiled molecule so that the plasmid remains circular,
but now assumes the relaxed conforma7on as all of the supercoiling is unraveled.
The amount of relaxed plasmid DNA from cell lysates seen in agarose gels depends
on the severity of the isola7on procedure and on the size of the plasmid (larger
plasmids will be more likely to be nicked) and can vary from a few % to about 50% of
the plasmid yield. The footnotes to both gel profiles above are very important! The
number of DNA molecules of DNA in each band in the leE gel panel of linear
standards is the same. By analogy, say you take a bundle of very long spaghew (say
100 strands) and you break it progressively into shorter and shorter sec7ons (like the
5 standards above). Each sec7on of spaghew – though of different lengths – will have
the same number of strands in the bundle!

35
Iden7fying linear fragments from plasmid digests can be difficult, but is much easier
when you take into account the number of resultant linear fragments and their
apparent sizes and posi7ons in an agarose gel aEer the plasmid has been cut or
digested with a restric:on enzyme (later lecture). For example, if the two plasmid
bands in the leE gel (supercoiled and relaxed, but of the same plasmid) are cut at a
single site, then a single linear band will result and this band MUST run at a posi7on
higher in the gel than the supercoiled band, but lower than the relaxed circular
plasmid band (check back to slides 9 & 10). In the example depicted on the right, this
7me with the same plasmids in the leE panel gel cut at two different sites, there will
be two linear fragments, but only one set (Lane 1 or Lane 2) shown in the gel on the
right can be correct. Which is it and why?

36