Chapter 10 | DNA Structure and Analysis

Phosphodiester Bond Formation

Two nucleotides join through dehydration synthesis, linking the phosphate of one nucleotide to the 3’ carbon of another sugar

Ligase function in NHEJ

DNA ligase regenerates phosphodiester bonds by connecting a monophosphate to a base using ATP hydrolysis (a nonspontaneous, exergonic reaction)

Energy of phosphodiester bond formation

Formation of the bond between the OH of the phosphate and the 4' carbon is endergonic (requires energy input)

Sugar in a nucleotide

A 5-carbon sugar where:

• 1′ carbon attaches to base via a glycosidic linkage,

• 2′ carbon has OH (ribose) or H (deoxyribose),

• 3′ and 5′ carbons allow DNA chain formation,

• A missing 4′ OH prevents phosphodiester bonding

Chargaff’s findings

• A = T, G = C

• A + T ≠ G + C

• A + G = C + T → purines = pyrimidines

• Ratios consistent across all organisms

DNA double helix structure

DNA forms a right-handed double helix with a phosphate backbone outside and bases inside held by hydrogen bonds

DNA backbone

Negatively charged and hydrophilic, facing the aqueous environment

Base orientation

Bases are hydrophobic and face inward, pairing via hydrogen bonds (A–T, G–C)

Antiparallel strands

Two DNA strands run in opposite directions (5′→3′ vs 3′→5′)

Helix dimensions

• 1 base pair = 3.4 Å apart

• 10 base pairs per turn (~34 Å)

• Diameter = 20 Å constant due to purine–pyrimidine pairing

DNA grooves

Alternating major and minor grooves where proteins interact noncovalently (H-bonds, ionic, van der Waals); major groove provides more information

Base pairing rule

Purines (A, G) pair with pyrimidines (T, C) to maintain uniform diameter

A-T pairing

A (purine) pairs with T (pyrimidine) via 2 hydrogen bonds

G-C pairing

G (purine) pairs with C (pyrimidine) via 3 hydrogen bonds, making the pair more stable

Hyperchromic shift

Increase in UV absorbance (260 nm) as DNA transitions from double-stranded to single-stranded during heating

Cause of increased absorbance

In single-stranded DNA, nitrogenous bases are more exposed to UV light, increasing absorption

DNA melting temperature (T_m)

Temperature where half of DNA is denatured (≈77 °C for typical DNA)

Base composition and melting point

GC-rich DNA requires higher temperature (≈83 °C) to melt due to 3 H-bonds per base pair, compared to 2 in AT pairs

Pyrimidine

Nitrogenous base with a single-ring structure; includes cytosine, uracil, and thymine

Purine

Nitrogenous base with a double-ring structure; includes guanine and adenine

Nucleoside

A molecule consisting of a nitrogenous base bonded to a sugar, lacking a phosphate group

Phosphate labeling (α, β, γ)

Describes the order of phosphate groups: α (innermost), β (middle), γ (outermost)

Griffith’s Experiment

Discovered transformation: non-virulent rough Streptococcus pneumoniae could become virulent when mixed with heat-killed smooth cells

Avery Experiment

Demonstrated that DNA is the transforming principle by selectively destroying macromolecules; transformation ceased only when DNA was degraded

Hershey-Chase Experiment

Used bacteriophages labeled with radioactive sulfur (proteins) and phosphorus (DNA) to show that DNA, not protein, enters bacterial cells during infection

Guthrie-Sinsheimer Experiment

Used isolated DNA from Phi-X174 phage to infect E. coli and produce new viral particles, confirming DNA alone can direct replication

Modern evidence for DNA as genetic material

Genetic engineering experiments show DNA from one organism can be inserted into another to produce new proteins

Molecular complementarity

Ability of nitrogenous bases to pair specifically (A with T/U, G with C)

Hybridization

Formation of double-stranded structures from complementary DNA and RNA strands

PCR components

Template DNA, primers, dNTPs, DNA polymerase, and buffer

Denaturation (PCR)

Heating to 95 °C separates DNA strands

Annealing (PCR)

Cooling to ~55 °C allows primers to bind to template DNA; temperature is ~5 °C below Tₘ

Extension (PCR)

DNA polymerase synthesizes new DNA strands from primers

PCR cycle

Repetition of denaturation, annealing, and extension (~35 times) leads to exponential DNA amplification

Restriction endonuclease

Enzyme that cuts DNA at specific internal sites; part of bacterial defense against viruses

Methylation in bacteria

Protects host DNA from restriction enzyme digestion

Sticky ends

Overhanging single-stranded ends formed by staggered DNA cuts, allowing complementary base pairing

Blunt ends

Straight cuts across both strands without overhangs; do not base-pair spontaneously

DNA ligase

Enzyme that reconnects DNA fragments cut by restriction enzymes

Multiple cloning site (MCS) / Polylinker

Region in a plasmid with many unique restriction sites for inserting DNA fragments