DNA structure- Part II

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• DNA structure- Part II

Levels of DNA structure
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Levels of DNA structure

• 1structure the order of bases on the
  polynucleotide sequence the order of bases
  specifies the genetic code.
• 2structure the three-dimensional conformation
  of the polynucleotide backbone.
• 3structure supercoiling.
• 4structure interaction between DNA and proteins

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DNA - 1 Structure

• Deoxyribonucleic acids (DNA) is a biopolymer that
  consists of a backbone of alternating units of
  2-deoxy-D-ribose and phosphate.

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This bond which indicates that there are two
covalent bonds formed between the -OH and the
acidic phosphate group

• The 3-OH of one 2-deoxy-D-ribose is joined to
  the 5-OH of the next 2-deoxy-D-ribose with bases
  by a phosphodiester bond.

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• So, the Primary Structure is the sequence of
  bases along the pentose-phosphodiester backbone
  of a DNA molecule
• Base sequence is read from the 5 end to the 3
  end.
• System of notation single letter (A,G,C,U and T).

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• The backbone of DNA RNA is hydrophilic.
• The hydroxyl gp (OH) of the sugar residues form
  hydrogen bonds with water.
• The Phosphate gp, with pka ? 2, are completely
  ionized and vely charged at pH 7.
• The ve charges are neutralized by ionic
  interactions with ve charges on proteins, metal
  ions.
• A short nucleic acid containing 50 or fewer
  nucleotides is called an oligonucleotide, a
  longer is a polynucleotide.

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the 2 hydroxyl is absent in DNA
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Abbreviations of Nucleic Acid Sequences

• pA-C-G-T-AOH
• or pApCpGpTpA
• or pACGTA

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Nucleotide Bases affect the three dimensional
structure of nucleic acids

• The purine and pyrmidine bases are conjugated
  ring system.
• One result is that pyrmidines are planar
  molecules and purines are nearly planar with a
  slight pucker.
• Free purine and pyrimidine bases can exist in
  two or more forms called tautomers, depending on
  pH.

A tautomeric shift occurs when a proton changes
its position, resulting in a rare tautomeric form.
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• Standard and anomalous base-pairing arrangements
  that occur if bases are in the rare tautomeric
  forms. Base mispairings due to tautomeric shifts
  were originally thought to be a major source of
  errors in replication.
• Such structures have not been detected in DNA,
  and most evidence now suggests that other types
  of anomalous pairings are responsible for
  replication errors.

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• As a result of resonance, delocalized electrons
  in the conjugated ring rings are available to
  absorb UV light at 260nm.
• The chemical properties of the purine and
  pyrimidines give rise to two modes of interaction
  between bases
• Hydrophobic stacking, the bases relatively
  insoluble in water at neutral pH.
• Base pairing which result from H-bonding .

As a result of resonance, all nucleotide bases
absorb UV light.
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Nucleotides play additional roles in cells

• Adenosine is a building block for some important
  enzyme cofactor, such as NAD and FAD. The
  presence of an adenosine component in a variety
  of cofactors enables recognition by enzymes that
  share common structural features.

NAD
FAD
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• cAMP formed from ATP in a reaction catalyzed by
  adenylyl cyclase, is a common second messenger
  produced in response to hormones and other
  chemical signals

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DNA - 2 Structure

• Secondary structure is the ordered arrangement of
  nucleic acid strands.
• Double helix is a type of 2 structure of DNA
  molecules in which two anti-parallel
  polynucleotide strands are coiled in a
  right-handed manner about the same axis.
• Structure based on X-Ray crystallography.
• The molecule is stabilized by hydrophobic
  interactions in its interior and by hydrogen
  bonding between the complementary bases pairs A-T
  and G-C

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Base Pairing

• Base pairing is complementary.
• A major factor stabilizing the double helix is
  base pairing by hydrogen bonding between T-A and
  between C-G
• T-A base pair comprised of 2 hydrogen bonds.
• G-C base pair comprised of 3 hydrogen bonds

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Base stacking

• Bases are hydrophobic and interact by van der
  Waals interactions.
• In standard B-DNA, each base rotated by 32
  compared to the next and, while this is perfect
  for maximum base pairing, it is not optimal for
  maximum overlap of bases in addition, bases
  exposed to the minor groove come in contact with
  water.
• Many bases adopt a propeller-twist in which base
  pairing distances are less optimal but base
  stacking is more optimal and water is eliminated
  from minor groove contacts.
• The propeller twist of the base pairs results in
  purine-purine clash in the center of the helix.
  Because the purines are larger than the
  pyrimidine rings, they extend beyond the helical
  axis of DNA.

Hydrophobic, van der Waals, and electrostatic
interactions favor the alignment of bases in an
aqueous solution or within a polynucleotide chain
the un-stacked orientation is disfavored.
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DNA attempts to reduce purine-purine clash in
several ways

• A. The base pairs rotate less along the helix
  axis in the purine-pyrimidine
• sequences (lower average helical twist).
• They tend to rotate less in the
  pyrimidine-purine sequences (lower than average
  helical twist).
• The average helical twist was still very
  close to the 36 proposed by Watson and Crick.
• Another way DNA minimizes the purine-purine clash
  is that it bends toward the minor grove or major
  groove to reduce the interaction.
• C. Finally clashing base pairs could slide left
  or right toward the phosphodiester backbones to
  minimize the purine-purine interaction.

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Major and minor grooves

• The major groove is large enough to accommodate
  an alpha helix from a protein.
• Regulatory proteins (transcription factors) can
  recognize the pattern of bases and H-bonding
  possibilities in the major groove.

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DNA adopts different helical forms

• Nucleic acids are inherently flexible molecules.
  Numerous bonds in the sugar-phosphate backbone
  can rotate, and thermal fluctuation can lead to
  bending, stretching, and un-pairing of the two
  strands.
• As a result, cellular DNA contains significant
  deviations from the Watson-Crick DNA structure,
  some or all of which may play important roles in
  DNA metabolism.
• Generally, such structural variations do not
  affect the key properties of strand
  complementarity antiparallel strands and the
  requirement for AT and GC base pairs.

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Variation in the three-dimensional structure of
DNA reflect three things
(1) The different possible confirmations of the
deoxyribose.
(2) Rotation about the contiguous bonds that make
up the sugar-phosphate backbone.
(3) Free rotation about the glycosidic bond.
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• B-DNA (Watson-Crick Form)
• considered the physiological form
• a right-handed helix, diameter 11Å
• 10 base pairs per turn (34Å) of the helix.
• A-DNA (favored in environment with very
• low water content)
• a right-handed helix, but thicker than
  B-DNA.
• 11 base pairs per turn of the helix
• has not been found in vivo.
• Z-DNA occurs at high salt concentrations in
• polymers that have a sequence of
  alternating purines and pyrimidines
• a left-handed double helix.
• may play a role in gene expression.
• Z-DNA occurs in nature, usually consists of
  alternating purine-pyrimidine bases.
• Methylated cytosine found also in Z-DNA.

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In each case, the sugar-phosphate backbones wind
around the exterior of the helix (red and blue),
with the bases pointing inward. The same
25-base-pair DNA sequence is shown in all three
forms. Differences in helical diameter can be
seen in end-on views (top) differences in
helical rise and groove shape are apparent in the
side views (bottom). B-DNA, the most common form
in cells, has a wide major groove and a narrow
minor groove. A-form helices, common for RNA and
certain DNA structures, are more compact than
B-DNA. The major groove is deeper and the minor
groove is shallower than in B-DNA. Z-DNA, which
forms only under high salt conditions or with
CG-rich DNA sequences, is left-handed, and its
backbone has a zigzag pattern. It is less compact
than B-DNA, with a very shallow major groove and
a narrow and deep minor groove.
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DNA - 3 Structure

• Tertiary structure is the three-dimensional
  arrangement of all atoms of a nucleic acid
  commonly referred to as supercoiling.
• The term "supercoiling" means literally the
  coiling of a coil.
• DNA is coiled in the form of a double helix.
• Enzymes called topoisomerases or gyrases can
  introduce or remove supercoils.
• If there is no net bending of the DNA axis upon
  itself, the DNA is said to be in
  a relaxed state. 
• Negative supercoiling may promote cruciforms

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Certain DNA Sequences Adopt Unusual Structures

• Other sequence-specific DNA structures have been
  detected, within larger chromosomes, that may
  affect the function and metabolism of the DNA
  segments in their immediate vicinity.
• For example, certain repetitive sequences can
  bend the DNA helix in a distinct way.
• This DNA bending helps certain proteinssuch as
  transcription factors, which promote the
  synthesis of mRNAsbind to their target DNA
  binding sites.
• Regions of DNA where the two complementary
  strands have the same sequence when read in the
  5'?3' or the 3'?5' direction occur relatively
  frequently in chromosomal DNA and are called
  palindromes.

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• In language, a palindrome is a word, phrase, or
  sentence that is spelled identically when read
  either forward or backward.
• Two examples are ROTATOR and NURSES RUN.
• In biology, the term applies to double-stranded
  regions of DNA where one strands sequence is
  identical to its complement.
• for example, 5'-GAATTC-3' is a palindrome
  because its complementary sequence is also
  5'-GAATTC-3'.
• Palindromes are formed from adjacent inverted
  repeats, which can occur within one strand of DNA
  or over the two strands of the double helix

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• These sequences play important biological roles,
  such as
• Slowing or blocking protein synthesis by the
  ribosome-a process called translation attenuation
  .
• Forming recognition sites for restriction
  enzymes, which catalyze double-stranded DNA
  cleavage.

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DNA classes and sizes
Circular DNA is a type of double-stranded DNA in
which the 5 and 3 ends of each stand are joined
by phosphodiester bonds.
? phage DNA
Human chromosome DNA
Plasmid DNA
E. Coli DNA
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Gene
What is in the middle??????
Something Happens
Protein
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RNA Structure

• In the early 1970s, Alexander Rich, Aaron Klug,
  and Sung-Hou Kim independently solved the
  structures of transfer RNAs, revealing how tRNAs
  carry the amino acids that are used in protein
  synthesis on the ribosomes.
• The wide-ranging functions of RNA reflect a
  structural diversity much richer than that
  observed in DNA molecules.

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• The single strand of RNA folds back on itself to
  form short base-paired or partially base-paired
  segments connected by unpaired regions.
• This property, called RNA secondary structure,
  enables RNA molecules to fold into many different
  shapes that lend themselves to many different
  biological functions.

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• The greater structural variety in RNA relative to
  DNA reflects the three main chemical differences
  between the two polynucleotides
• 1- The pentose (2'-deoxyribose in DNA vs. ribose
  in RNA).
• 2- The base composition (thymidine vs. uridine).
• 3- The sugar pucker of the pentose (C-2' endo vs.
  C-3' endo).
• Double-stranded RNAs do exist in nature, such as
  those that form the genomes of some viruses.
• In addition, some RNAs do not seem to form stable
  three-dimensional structures from local
  base-pairing interactions, e.g. mRNA.
• These RNAs may fold into three-dimensional
  structures only in the presence of bound
  proteins, forming complexes called
  ribonucleoproteins (RNPs).

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RNAs Form Various Stable Three-Dimensional
Structures

• Most of the highly structured RNAs contain
  noncanonical base pairs and backbone
  conformations not observed in DNA.
• In many cases, the 2'-hydroxyl group on ribose, a
  chemical feature that distinguishes RNA from DNA,
  seems to be directly or indirectly responsible
  for these unique structural properties.
• The presence of the 2' hydroxyl makes RNA
  vulnerable to hydrolysis, but it also allows for
  additional hydrogen bonding between segments of
  an RNA molecule.
• As a result, RNA helices are more
  thermodynamically stable than are DNA helices of
  the same length and sequence.

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• Base pairs other than canonical AU and CG pairs
  are common in RNAs, including A-A and G-U. In all
  cases, base pairs or single bases are most stable
  when stacked on top of one another in a helix.
• Divalent and monovalent metal ions (Mg2, Ca2,
  K, and Na) bind to specific sites in RNA and
  help shield the negative charge of the backbone,
  allowing parts of the molecule to pack more
  tightly together.

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RNA is very similar to DNA
Chemically, RNA is very similar to DNA. There are
some main differences 1- RNA uses the sugar
ribose instead of deoxyribose in its backbone.

• Vicinal Hydroxyl Group Makes Difference !!
• The vicinal OH groups of RNA are susceptible to
  nucleophilic attach leading to hydrolysis of the
  phosphodiester bond.
• DNA is not susceptible to alkaline hydrolysis.
  RNA is alkali labile.

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2- RNA uses the base Uracil (U) instead of
Thymine (T). U is also complementary to A.
But Why DNA Contains Thymine rather than
Uracil?!

• It is because cytosine deaminates to form uracil
  in a finite rate in vivo. This would results in a
  mutation in the DNA
• So any U found in DNA will be corrected by a
  proofreads system. Thus DNA can not normally
  have U.

Guanine and cytosine form a base pair stabilised
by three hydrogen bonds, whereas adenine and
thymine bind to each other through two hydrogen
bonds. The red frames highlight the functional
groups of cytosine and thymine that are
responsible for forming the hydrogen bonds.
Cytosine can spontaneously undergo hydrolytic
deamination, resulting in a uracil base with the
same capability for hydrogen bond formation as
thymine. 
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• 3- RNA tends to be single-stranded.
• 4- Functional differences between RNA and DNA.
• DNA single function, RNA many functions
  according to their type.
• Example of types of RNA e.g tRNA, mRNA, rRNA.

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The Central Dogma

• The Flow of Information DNA ? RNA ? Protein.
• A gene is expressed in two steps
• DNA is transcribed to RNA.
• Then RNA is translated into protein.

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Many RNA molecules do not encode proteins

• tRNA.
• rRNA.
• a vast number of other non-coding RNAs (ncRNAs).

- 98 of the human genome does not code for
protein. What is its function?
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How big part of human transcribed RNAresults in
proteins?

• Of all RNA, transcribed in higher eukaryotes,
  98 are never translated into proteins.
• Of those 98, about 50-70 are introns.
• 4 of total RNA is made of coding RNA.
• The rest originate from non-protein genes,
  including rRNA, tRNA and a vast number of other
  non-coding RNAs (ncRNAs).
• Even introns have been shown to contain ncRNAs,
  for example snRNAs.
• It is thought that there might be order of
  10,000 different ncRNAs in mammalian genome

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The Other 98 of the Human Genome

• ncRNA genes have diverse and essential roles.
• May be relics of ancient RNA-based life.
• Many cellular machines contain RNA.
• Ribosome rRNA
• Spliceosome snRNAs (U1,U2,U4,U5,U6)
• Telomerase Telomerase RNA

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RNA types
Non-coding RNA. Transcribed RNA with a
structural, functional or catalytic role
mRNA
rRNA Participate in protein synthesis
snRNA found in nucleolus, involved in
modification of rRNA.
tRNA Interface between mRNA amino acids
snRNA RNA that form part of the spliceosome
miRNA involved regulation of expression
Other large RNA with roles in chromotin structure
stRNA- Small temporal RNA, with a role
in developmental timing
siRNA- Small interfering RNA, Active molecules
in RNA interference
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• RNA molecules are classified according to their
  structure and function

Kinds of RNA The Role of Different Kinds of RNA The Role of Different Kinds of RNA The Role of Different
Function Size RNA Type
Transport amino acids to site of protein synthesis Small tRNA
Combines with proteins to form ribosomes, the site of protein synthesis Variable size rRNA
Direct amino acid sequence of proteins. Variable mRNA
mRNA to it is mature form in eukaryotes Processes initial Small snRNA
Affect gene expression Small siRNA
Affect gene expression Small miRNA
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tRNA

• The smallest kind of the three RNAs.
• A single-stranded polynucleotide chain between
  73-94 nucleotide residues.
• Intramolecular hydrogen bonding occurs in tRNA.

• tRNA is a cloverleaf shaped, single strand.
• It has
• Anticodon loop.
• Amino acid binding site at the 3end.
• Two other loops for binding the ribosomes.

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rRNA

• rRNA a ribonucleic acid found in ribosomes, the
  site of protein synthesis.
• Only a few types of rRNA exist in cells.
• Ribosomes consist of 60 to 65 rRNA and 35 to
  40 protein.
• In both prokaryotes and eukaryotes, ribosomes
  consist of two subunits, one larger than the
  other.

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mRNA

• A ribonucleic acid that carries coded genetic
  information from DNA to ribosomes for the
  synthesis of proteins.
• Present in cells in relatively small amounts and
  has a short-half life.

• Single stranded and unstable.
• Biosynthesis is directed by information encoded
  on DNA.
• A complementary strand of mRNA is synthesized
  along one strand of an unwound DNA, starting from
  the 3 end.

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Eukaryotic mRNA Structure

• Capping linkage of 7-methylguanosine 7 to the 5
  terminal residue.
• Tailing attachment of an adenylate polymer (poly
  A)

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Chemical and thermodynamic importance of the DNA
structure
Denaturation and renaturation
Complementary base paring by hydrogen bonding and
van der Waals interaction ? storage and transfer
of genetic information
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Properties of DNA- Denaturation

• When DNA is heated to 80OC, its UV absorbance
  increases by 30-40 .
• With heating, noncovalent forces holding DNA
  strands together weaken and break.
• When the forces break, the two strands come apart
  in denaturation or melting.

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Denaturation of DNA

• As strands separate, absorbance at 260 nm
  increases.
• Stacked base pairs in native DNA absorb less
  light .
• Increase is called hyperchromicity.
• Temperature at which DNA strands are ½ denatured
  is the melting temperature or Tm.
• Melting Temperature (Tm) the temp. at which
  half of the helical structure is lost

- What is the hyperchromic shift
Hyperchromic Effect a large increase in light
absorbance at 260 nm occurring as double-helical
DNA is melted (i.e. unwound).
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Melting temperature and G-C

• GC content of DNA has a significant effect on Tm
  with higher GC content meaning higher Tm.

Examples

• Upon denaturation, the H-bonds between the base
  pairs of a native nucleic acid are replaced by
  energetically equivalent hydrogen bonds between
  the bases and water

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• In addition to heat, DNA can be denatured by
• Organic solvents.
• High pH.
• Low salt concentration.
• GC content also affects DNA density
• Direct, linear relationship

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DNA Renaturation

• After two DNA strands separate, under proper
  conditions the strands can come back together
• Process is called annealing or renaturation
• Three most important factors
• Temperature best at about 25 C below Tm.
• DNA Concentration within limits higher
  concentration better likelihood that 2
  complementary will find each other.
• Renaturation Time as increase time, more
  annealing will occur

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Summary

• GC content of a natural DNA can vary from less
  than 25 to almost 75.
• GC content has a strong effect on physical
  properties that increase linearly with GC
  content.
• Melting temperature, the temperature at which the
  two strands are half-dissociated or denatured.
• Density.
• Low ionic strength, high pH and organic solvents
  also promote DNA denaturation.

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Hybridization
For What
Annealing of complementary DNA (hybrid duplex)
from different species at 65?.
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• Hybridization is a process of putting together a
  combination of two different nucleic acids.
• Strands could be 1 DNA and 1 RNA.
• Also could be 2 DNA with complementary or nearly
  complementary sequences.

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DNA Sizes

• DNA size is expressed in 3 different ways
• Number of base pairs .
• Molecular weight 660 is molecular weight of 1
  base pair.
• Length 33.2 Å per helical turn of 10.4 base
  pairs.
• Measure DNA size either using electron
  microscopy or gel electrophoresis.

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Summary

• Natural DNAs come in sizes ranging from several
  kilobases to thousands of megabases.
• The size of a small DNA can be estimated by
  electron microscopy.
• This technique can also reveal whether a DNA is
  circular or linear and whether it is supercoiled.