Chapter 10 - DNA Structure and Analysis

Learning goals

Understand and be able to describe the experiments that determined that DNA is the hereditary material:

• Griffiths; Avery, MacLeod and McCarty; Hershey-Chase; Watson and Crick.

Understand how DNA structure affects its function:

• Be able to draw the sugar-phosphate backbone and each nucleotide; be able to find the 5’ and 3’ ends of the chains

• Understand the consequences of the antiparallel nature of the duplex.

• Be able to find where the hydrogen bonds are between base pairs; understand the differences in the chemical nature of the ends (i.e., the phosphate group(s) and the 3’ hydroxyl).

For a molecule to serve as the genetic material, it must be able to:

• Be replicated (copied) and distributed to daughter cells

• Store the molecular information (protect it, repair it)

• Express the information

• Allow variation by mutation

Until 1944, observations favored protein as the genetic material

• Proteins and nucleic acids were the major candidates for the genetic material

• For a long time, protein was favored to be the genetic material

  • It is abundant in cells

  • It was the subject of the most active areas of genetic research

  • DNA was thought to be too simple to be the genetic material, with only four types of nucleotides as compared to the 20 different animo acids of proteins

Frederick Griffithc

• British physician and bacteriologist during WWI

  • He assayed the pneumococci samples taken from patients and classified them to study the pneumonia epidemiology

  • He experimented on mice to understand the pathology

• Credited as the first to show that the bacterium can change

• Died in an air raid in London

Transformation by Griffith

• Showed that avirulent strains of the bacterium Diplococcus pneumoniae could be transformed to virulence

  • The term for bacterial transformation came from this experiment

  • He speculated that the transforming principle could be part of the polysaccharide capsule or some compound required for capsule synthesis

Avery, MacLeod, & McCarty

• Followed Griffith’s experiment

• Demonstrated that the transforming principle was DNA, not protein

• This work was recognized by some but ignored by many scientists

• It was not recognized by the Nobel committee, which later apologized

Hershey-Chase Experiment

• The work on DNA as the molecule of heredity was followed by experiments from the “Phage Church” group

• Hershey and his technician Chase performed a critical experiment to demonstrate that DNA is the molecule of heredity

  • Transfection of viral DNA into bacterial cells proved conclusively that the viral DNA alone contains all the necessary information for production of mature viruses

Evidence for DNA as the genetic molecule

• Indirect evidence

  • DNA is found only where the primary genetic function occurs, whereas protein is found throughout the cell

  • Correlation of UV light absorption and mutagenesis

    • UV light is capable of inducing mutations in the genetic material and is most mutagenic at a wavelength of 260 nm.

    • DNA and RNA absorb UV light most strongly at 260 nm, but protein absorbs most strongly at 280 nm, a wavelength at which no significant mutagenic effects are observed.

• The strongest direct evidence for DNA as the genetic material comes from recombinant DNA technology.

  • Recomb. DNA tech. is the expression of eukaryotic genes in bacteria

    • e.g., making a copy of the human insulin gene (cloning), expressing it in bacteria, and recovering the produced hormone from the growth medium.

  • The presence of eukaryotic DNA and protein products produced from it are strong direct evidence that DNA is the material of heredity

DNA Structure, KNOW

• nucleic acid chemistry

• DNA structure which is essential to understand its functions

Nucleotides

• Are the building blocks of nucleic acids

  • They are assembled into polynucleotides of DNA and RNA

• They consist of

  • A nitrogenous base

  • A pentose sugar (5 carbon)

  • A phosphate group

Nitrogenous bases can be purines or pyrimidines; they are heterocyclic rings

Purines and pyrimidines are grouped into those with keto bases (G, T, U) or amino bases (A, C)

A sugar can be

Ribose or deoxyribose

Nucleoside

contains the nitrogenous base and the pentose sugar

Nucleotide

is a nucleoside with a phosphate group added

Nucleotides can have

one, two, or three phosphate groups and are called NMPs, NDP, and NTPs, respectively

RNA contains

ribose as its sugar

DNA contains

deoxyribose as its sugar

The C-5’ position is the location of the phosphate group on a nucleotide

• There can be one, two, or three phosphates

  • Ex. NMP, AMP, ADP, ATP

DNA and RNA both contain A, C, G

Only DNA contains T

Only RNA contains U

Nomenclature

Phosphodiester bonds

• Nucleotides are linked by a phosphodiester bond between the phosphate group at the C-5’ position and the OH group on the C-3’ position

  • An ester is -C-O-C

  • A phosphoester is -C-O-P-

  • Phosphodiesters have two adjacent phosphoester bonds; the phosphorus is shared

Chargaff showed

that the amount of A is proportional to T and the amount of C is proportional to G, but the percentage of C+G does not necessarily equal the percentage of A + T

X-ray diffraction of DNA showed

a 3.4 angstrom periodicity, characteristic of a helical structure

• This was Rosalind Franklin’s work

Watson and Crick

• proposed DNA is a right-handed double helix in which the two strands are antiparallel and the bases are stacked on one another

• They built their model based on

  • Chargaff’s findings

  • Chemical principles

    • Bond angles and types

    • Solvent (water) interactions

    • Stacking, pi-bond electrons

  • Franklin’s diffraction data (A and B forms)

    • B form: 34 angstron periodicity

  • Modeling: cardboard cut-outs and trial and error

• The two strands are connected by A-T and G-C base pairing and there are 10 base pairs per helix turn

B form DNA

The Watson-Crick DNA model is of B-DNA, which is the biologically significant form

DNA base pairs are tightly stacked

• Like pancakes

• Highly regular spacing

• Accounts for 3.4 Angstrom spots

  • (X-ray diffraction) due to pi-bond interactions

• No room for water or ions

Phosphates are far apart

• Alternating sugars (gray and white) and phosphates (orange and red)

• Sugar-phosphate backbone fully extended

• Phosphates are negatively charged - charge repulsion

Base complementarily due to hydrogen bonding

• Provides complementarily of the two strands

• Explains Chargaff’s rule

• Provides chemical stability to the helix

  • Partial positive (amines) and negative charges (keto oxygen)

  • A-T base pairs form two hydrogen bonds; G-C base pairs form three hydrogen bonds

• Base pairs have the same dimension provided a uniform width to the double helix

Base pairs lead to uniform helix width

• DNA seen “end on” view

• Base pairs (grey and blue) form uniform chentral column

• Sugar-phosphate backbone (orange and red) wraps around the outside at a uniform distance ~20 angstroms

Base-pair edges are each unique

Provides a mechanism for regulatory proteins to bind to specific DNA sequences without separating the strands

Other conformations of DNA

• Alternative forms of DNA exist

  • A-DNA is slightly more compact than B-DNA

    • Double-stranded RNA and RNA-DNA hybrids are in A-form

  • Z-DNA forms a left-handed double helix

Comparison of A- and B-DNA

Other DNA structures

RNA

• the sugar ribose replaces deoxyribose

• and uracil replaces thymine of DNA

Most RNA is thought of as single stranded, but…

• RNAs form double-stranded regions as they fold into different secondary structures; these double-stranded regions can be critical for function

Classes of RNA

• All RNAs originate as complementary copies of one of the two strands of DNA

  • DNA serves as the template for their synthesis (transcription is synthesis of RNA from DNA)

• There are four classes of cellular RNAs (at least four, depending on how they’re grouped):

  • messenger RNA (mRNA)

  • ribosomal RNA (rRNA)

  • transfer RNA (tRNA)

  • other RNAs: snRNA, snoRNA, siRNA, miRNA, IncRNA

Structure of RNA

• Most RNA is thought of as single stranded, but

  • RNAs form double-stranded regions as they fold into different secondary structures; these double-stranded regions can be critical for function

Learning Goals

• Be able to recognize and distinguish the nucleotides from each other; be able to draw them

• Understand that there are different shapes of DNA beyond B-form

• Understand the key differences between RNA and DNA

• Understand how the chemistry & biochemistry of nucleic acids are used in the analysis of the molecule

Van der Waals forces

Weak, distance-dependent electrostatic attractions that occur between neutral atoms or molecules rather than holding individual atoms together inside a single molecule

The current thinking is that RNA was an early evolving molecule that could self-replicate and do catalysis:

• It preceded a world dependent on DNA and proteins

• Remnants of this world are seen in the ribosome, ribozymes, splicing machinery, reverse transcriptase

U vs T

• Thymine is also known as 5-methyl uracil

• In biosynthetic pathways, T is made from U

  • → UMP → dUMP → dTMP

• These suggest that T is derived, and U was present initially

U can form base pairs with…

• Watson-Crick A-U base pair

• Hoogstein A-U base pair

• G-U base pair

• U-U base pair

• U is the most promiscuous of bases: it forms non-Watson-Crick base pairs

• If U pairs with something other than A, this can lead to a mutation (change) in the sequence

• The methyl group in T protrudes into the major groove and helps to stabilize A-T base pairs over other pairing options

  • Watson-Crick A-U base pair

  • The methyl group in T increases fidelity during DNA replication

C deaminates to U

• C spontaneously undergoes deamination (hydrolysis reaction) to become U

• If U was in DNA and this deamination occurred, how could cells distinguish a real U from a deaminated C?

  • Instead, cells use T (methyl-U) to base pair with A

  • This means that when U occurs in DNA (arising from deamination of C), it can be recognized and removed

Why is there U but not T in RNA

Change to: why have T in DNA instead of U

Increased fidelity during replication and decreased mutation rate (DNA repair will remove U)

RNA chains are more susceptible to cleavage than DNA chains

• In alkaline solutions, the 2’ hydroxyl loses a proton; it then acts as a nucleophile to attack the phosphorus atom of the phosphoester linkage, breaking the chain.

• DNA lacks the 2’-OH and cannot do this reaction.

Why is there ribose in RNA?

Change to: why is there deoxyribose in DNA?

Increased stability of the DNA chains

Analytical techniques to investigate DNA and RNA

• Spectroscopy

• Denaturation and renaturation of the double helix

  • Hybridization of molecules, probes and primers: e.g., melting profiles; in situ hybridization (FISH - fluorescent in situ hybridization and non-fluorescent detections); primers in PCR reactions

• Electrophoresis: separation and detection

• Cleavage of DNA by restriction enzymes

• DNA sequence analysis

DNA/RNA analysis is based on their chemical properties

• The chemistry of the nucleic acids allows for analysis

  • Property 1: conjugated double bonds in the bases

    • Absorption of UV light at 260 nm; spectroscopy (A260)

  • Property 2: percent G+C, mass and shape

    • DNA molecules that differ in GC content have different buoyant densities (sedimentation equilibrium centrifugation)

    • Molecules with different masses and shapes move differently through gradients (sedimentation velocity centrifugation)

  • Property 3: hydrogen bond formation & stacking interactions

    • Denaturation/renaturation (percent G+C; Cot analysis; renaturation kinetics; denaturation in PCR)

  • Property 4: negative charge of the phosphates

    • Electrophoresis

  • Property 5: order of base pairs, i.e., their sequence

    • Denaturation/renaturation (“probing” in FISH, Southern)

Spectroscopy

• Nucleic acids absorb ultraviolet (UV) light most strongly at 260 nm due to interaction between UV light and the conjugated double bonds in the rings of the bases

• The absorption at 260 nm can be used to determine the concentration of nucleic acids in a solution

  • 1 A260 = 0.05 mg/ml of DNA

• Absorption by itself does not indicate the form of the molecule

  • single- vs. double-stranded; nucleotide vs. oligonucleotide vs. polynucleotide)

• The action spectrum is the wavelength of light that causes mutations.

  • The energy from UV light causes covalent bonds to form between adjacent pyrimidine bases.

• Nucleic acids absorb UV light most strongly at 260 nm

  • protein absorbs most strongly at 280 nm, due to absorption by tryptophan, tyrosine and phenylalanine

Centrifugation

• Nucleic acids can be separated by gradient centrifugation procedures.

  • Sedimentation equilibrium centrifugation separates by their buoyant density on a salt gradient.

    • Opposing forces: centrifugal force (sedimentation) vs. diffusion

    • A sensitive measure of density

• Sedimentation velocity centrifugation measures the velocity of sedimentation through a gradient; measurement is in Svedberg (S) coefficient units.

  • Opposing forces: viscosity (friction) and diffusion

  • Measure of density, mass and shape (bigger, heavier and more compact molecules sediment faster. E.g, the 80S ribosome or the 16S rRNA molecule.

Sedimentation velocity centrifugatino

• Samples can also be monitored in real time for their movement through a density gradient.

  • Detection occurs via absorption of UV light.

  • Movement through the gradient is a function of both mass and shape of the molecule.

  • Measured in Svedberg (S) units.

    • E.g., the 80S ribosome; the 18S rRNA molecule

Denaturation and renaturation

• Denaturation and renaturation can provide information about the composition and complexity of DNA samples.

  • Denaturation (melting) is the separation of DNA molecules into two strands.

    • Breaking of non-covalent bonds • dsDNA → ssDNA

  • Renaturation is the reforming of double stranded molecules from single strands; formation of base pairs.

    • Renaturation – generally used when the molecules are the same (i.e., both strands of DNA)

    • Annealing is used with oligonucleotides as probes or primers

    • Hybridization is synonymous with annealing, and originally came from forming double-stranded hybrid molecules between DNA and RNA

Misconception: denaturation is “broken”

Denature: to change the nature of

• In physics – the addition of material to block nuclear fission In chemistry – the addition of a noxious agent (MeOH) to EtOH to make denatured ethanol

• In biology – (a) to cause the structure of a protein to unfold so that its biological activity is diminished or eliminated; (b) to cause the paired strands of double-stranded DNA to separate into individual single strands.

• In both (a) and (b), non-covalent bonds, such as hydrogen bonds, are broken. Proteins typically cannot refold to restore their activity (too complex) but DNA can re-anneal because of specific base pairing.

Denaturation and renaturation

• Denaturation (melting) and renaturation (annealing or hybridization) by the formation of dsDNA.

  • Critical parameters include

    • For melting: The number of G-C base pairs, length of the molecules, salt concentration.

    • G-C content: Higher GC → more stable

    • Length: Longer → more stable

    • Salt concentration: Higher → more stable

  • For renaturation: The number of G-C base pairs; complexity of the sequences (repetitive or unique); length and concentration of the molecules.

• Applications (many important applications)

  • Melting temperature determination

  • primer binding for PCR

  • probe binding in hybridization (Southerns, northerns, microarray, FISH, gene expression studies, etc.)

  • reassociation (Cot) analysis

Melting temperature determination

• The temperature of the sample is raised and the absorption at 260 nm is followed.

  • Hyperchromic shift:

    • Single- stranded DNA absorbs more UV light than double stranded DNA

  • Melting temperature (Tm)

    • is when half of the molecule is denatured

  • Higher GC → higher Tm

    • GC rich DNA is more stable

Melting temperature (Tm) is affected by:

G-C content: Higher GC → more stable

Length: Longer → more stable

Salt concentration: Higher → more stable

Renaturation kinetics (Cot) is affected by:

G-C content: Higher GC → faster

Sequence complexity: Simpler → faster

Length: Shorter → faster

Concentration: Higher → faster

Reassociation kinetics

• provides information about the genome size and complexity of DNA from an organism

  • DNA is broken into pieces ~several hundred bp in length

  • Sample is heated to denature (dissociate into single strands) the DNA

  • Temperature is lowered

  • Reassociation into dsDNA is measured

Reassociation kinetics provides information about the size and complexity of genomic DNA

Melting temperature (Tm) is affected by:

• G-C content: Higher GC → more stable

• Length: longer → more stable

• salt concentration: higher → more stable

Renaturation kinetics (Cot) is affected by:

• G-C content: higher GC → faster

• Sequence complexity → simpler → faster

• Length: shorter → faster

• Concentration: higher → faster

Applications - many but most important:

• PCR primer binding

• Hybridization (Southerns, Northerns, microarray, FISH, gene expression studies, etc)

• Reassociation (Cot) analysis - DNA complexity

In molecular hybridization,

DNA strands (or DNA and RNA strands) can be annealed to each other

Fluorescent in situ hybridization (FISH) is used to

identify the chromosomal location of a DNA of interest

• The yellow color is due to hybridization of a probe that binds to (i.e., recognizes) the centromere; the probe is labeled with a molecule that fluoresces yellow.

• The red color is due to a dye (propidium iodide) that binds to DNA non- specifically.

FISH analysis to identify genes associated with disease

• Example of hybridization used to see a chromosome inversion found in a patient with Tourette’s syndrome.

  • The green and red fluorescent probes are lots of small DNA fragments that specifically bind to a larger DNA region

  • This was used to map a gene, SLITRK1, and associate it with the disorder.

Position of probes on the chromosome - inversion separates the region

Probes are a collection of small DNA molecules that can target specific DNA sequences. Thus they can cover (interrogate) large portions of the genome.

In situ hybridization

• is used to analyze gene expression, and uses a labeled complementary DNA, RNA, or modified nucleic acids strand as a probe) to localize a specific mRNA transcript in a section of tissue (in situ)

  • Probes are “labeled” with a radioactive isotope, a fluorescent molecule, or chemical stain.

  • The dark blue stain indicates where RNA is localized that is complementary to a probe for a maternal effect gene hunchback.

Polymerase chain reaction

• The Polymerase Chain Reaction Makes DNA Copies without Host Cells.

  • The polymerase chain reaction (PCR) copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities.

  • PCR requires two oligonucleotide primers that provide specificity and a heat stable DNA polymerase.

PCR

• PCR requires two oligonucleotide primers, one complementary to the 3' end of one strand of the DNA to be amplified and one complementary to the 3' end of the other strand. The primers anneal to denatured DNA, and the complementary strands are synthesized by a heat-stable DNA polymerase.

• The three steps of PCR — denaturation, primer annealing, and extension — are repeated over and over using a thermocycler to amplify the DNA exponentially.

PCR

DNA analysis is based on chemistry

• The chemistry of the nucleic acids allows its analysis

  • Property 1: conjugated double bonds in the bases

    • Absorption of UV light at 260 nm; spectroscopy (A260)

  • Property 2: percent G+C, mass and shape

    • DNA molecules that differ in GC content have different buoyant densities (sedimentation equilibrium centrifugation)

    • Molecules with different masses and shapes move differently through gradients (sedimentation equilibrium centrifugation)

  • Property 3: hydrogen bond formation

    • Denaturation/renaturation (percent G+C; Cot analysis; reassociation kinetics; denaturation in PCR)

  • Property 4: negative charge (phosphates) at neutral pH

    • Electrophoresis

  • Property 5: order of base pairs

    • Denaturation/renaturation (“probing” in FISH, Southern/Northern analysis, in situ analysis; primer binding in PCR)

    • Restriction digestion

    • DNA sequence analysis

Electrophoresis

• Nucleic acid electrophoresis separates DNA and RNA fragments by size such that smaller fragments migrate through a gel at a faster rate than large fragments

Restriction enzymes

• Restriction Enzymes Cut DNA at Specific Recognition Sequences.

  • A restriction enzyme is a endonuclease whose activity is restricted to a specific DNA recognition sequence.

  • DNA fragments are produced by DNA restriction.

• Most recognition sequences are palindromic, and restriction enzymes often cleave these sequences in an offset manner

Restriction enxyme cleavage sites are useful for DNA engineering

Some restriction enzymes cleave the sequences in an offset manner to leave bases that are not paired: these are called “sticky ends”

Restriction enzyme cleavage sites are useful for DNA engineering

Some restriction enzymes cleave both strands at the same place: these are called “blunt ends”.

Restriction enzyme cleavage sites are useful for DNA engineering

• Different Restriction enzymes can create the same sticky ends

• Restriction fragments with the same sticky ends can be “ligated” back together to create useful “recombinant” DNA molecules

• Can insert DNA into similarly cleaved plasmid “vectors” i.e. “cloning”

• Sometimes does not recreate the restriction site

Restriction and Electrophoresis

After separation, DNA or RNA fragments are visualized by staining with the dye ethidium bromide and examination under ultraviolet light

Sanger DNA sequencing

• The second method developed to sequence DNA sequencing is based on DNA synthesis.

• This is the dideoxy chain termination sequencing, developed by Fred Sanger.

  • This method is based on in vitro DNA synthesis.

  • The dideoxy nucleotides lack the hydroxyl group on both the 2’ and 3’ carbons.

  • A radioactive nucleotide is added to the sequencing reaction.

DNA synthesis

• Chain elongation occurs in the 5' to 3' direction by addition of one nucleotide at a time to the 3' end.

• As the nucleotide is added, the two terminal phosphates are cleaved off as the leaving group.

Dideoxy DNA sequencing

Autoradiogram examples

• Radioactivity is provided by the primer (or a small amount of a radionucleotide)

• Fragments are separated by electrophoresis using a gel system that can resolve band sizes by a single nucleotide.

• Each lane contains the reaction with a single dideoxy nucleotide with the 4 dNTPs (as shown on top, G means ddG was used).

• Read the sequence by starting at the bottom, which corresponds to the 5’ end closest to the primer.

Dideoxy sequencing

• Large-scale genome sequencing is automated and uses fluorescent dye-labeled dideoxynucleotides.

  • Each ddNTP is tagged with a different color fluorescent dye.

  • Each sample is size separated using capillary gel electrophoresis.

DNA sequence analysis

Example of sequence from automated machine

Massively parallel sequencing

• New DNA sequencing methods are being developed to be less expensive than the automated Sanger method.

• They use technologies that are “massively parallel”, i.e., millions of reactions performed on different samples in small scales at different places at the same time.

• Newest methods do not rely on the slowest steps associated with the Sanger method.

  • Sanger method: the cloning of fragments into plasmids and their recovery in bacteria.

  • Method from Illumina: generation of PCR products on glass slides

Illumina Sequencing

• PCR-like methods create localized clusters of millions of copies of the DNA on a glass slide. Each slide has many millions of different clusters

• Uses fluorescent dyes that act like reversible ddNTPs “blocking groups”

  • Add the base; record image of the fluorescence over the entire slide; remove the dye/blocking group; repeat hundreds of times; Computationally record/analyze the sequence at each spot

Next Generation Sequencing: Illumina

Illumina sequencing step

Illumina: new DNA sequencing methods are massively parallel

Advances in Sequencing Technology allowed >million-fold reduction in cost