Chapter 4 – Nucleic Acids

Learning Objectives

• Understand the nomenclature, structure, and features of bases, nucleosides, and nucleotides.

• Compare and contrast RNA and DNA.

• Describe the UV absorbance properties of DNA and how they differ from proteins.

• Explain the structures of DNA (A, B, Z forms) and the forces that stabilize them.

• Discuss DNA topology and agarose gel electrophoresis.

• Understand DNA denaturation and renaturation.

• Recognize palindromic DNA sequences, restriction enzymes, and basics of molecular cloning.

Nucleic Acids: General Features

Definition and Biological Importance

Nucleic acids are linear, polymeric molecules composed of nucleotide monomers. They store and transmit genetic information in all living organisms.

• The human genome contains approximately 3 billion base pairs.

• When fully extended, the DNA from a single human cell measures about 2 meters in length.

Bases, Nucleosides, and Nucleotides

Definitions and Nomenclature

• Bases: Nitrogenous bases are classified as purines (adenine, guanine) and pyrimidines (cytosine, thymine, uracil).

• Nucleoside: A base covalently linked to a sugar (ribose or deoxyribose).

• Nucleotide: A nucleoside with one or more phosphate groups attached, typically at the 5' position.

Example: Adenosine is a nucleoside; adenosine 5'-monophosphate (AMP) is a nucleotide.

Structural Differences Between DNA and RNA

• DNA contains deoxyribose sugar and the base thymine.

• RNA contains ribose sugar and the base uracil (instead of thymine).

• The 2'-OH group in RNA makes it more susceptible to hydrolysis, rendering DNA more stable for genetic storage.

UV Absorbance Features of DNA

Principles and Applications

• Both proteins and nucleic acids absorb ultraviolet (UV) light, but at different wavelengths.

• DNA and RNA absorb maximally at 260 nm, while proteins absorb at 280 nm.

• The ratio of absorbance at 260/280 nm is used to assess nucleic acid purity:

  • Pure DNA: 260/280 ≈ 1.8–2.0

  • Pure protein: 260/280 ≈ 0.5

Application: UV absorbance is used to quantify DNA and RNA in laboratory samples.

DNA Structures: A, B, and Z Forms

Double Helix and Stabilizing Forces

• B-DNA: The most common form in cells; right-handed helix with about 10.4 base pairs per turn.

• A-DNA: Right-handed, shorter and wider than B-DNA; forms under dehydrating conditions or in double-stranded RNA.

• Z-DNA: Left-handed helix; forms in sequences with alternating purines and pyrimidines (e.g., GC repeats).

• Stabilizing forces include hydrogen bonding between base pairs and base stacking (van der Waals interactions).

Base Pairing and Chargaff's Rules

• Watson-Crick base pairing: A pairs with T (or U in RNA), G pairs with C.

• Chargaff's rules: In double-stranded DNA, %A = %T and %G = %C.

DNA Topology

Supercoiling and Linking Number

• Supercoiling: The coiling of the DNA double helix upon itself, important for DNA compaction and regulation.

• Linking number (L): The total number of times one strand wraps around the other in closed circular DNA.

• Twist (T): Number of helical turns.

• Writhe (W): Number of superhelical turns.

• Relationship:

Example: Supercoiled DNA migrates faster in agarose gel electrophoresis than relaxed DNA.

Agarose Gel Electrophoresis

Principle and Interpretation

• DNA fragments are separated by size and conformation using an electric field in a porous agarose matrix.

• Negatively charged DNA migrates toward the anode (positive electrode).

• Supercoiled, linear, and nicked circular DNA migrate differently due to their shapes.

DNA Denaturation and Renaturation

Mechanisms and Effects

• Denaturation: The process by which double-stranded DNA unwinds and separates into single strands due to disruption of hydrogen bonds (by heat, alkali, or chemicals).

• Renaturation: The reformation of the double helix when denaturing conditions are removed.

• Hyperchromic effect: Denatured DNA absorbs more UV light at 260 nm than native DNA.

• DNA melting is cooperative, occurring over a narrow temperature range.

Palindromic DNA, Restriction Enzymes, and Molecular Cloning

Definitions and Applications

• Palindromic sequences: DNA sequences that read the same 5' to 3' on both strands; often recognition sites for restriction enzymes.

• Restriction enzymes: Proteins that cut DNA at specific palindromic sequences, essential tools in molecular cloning.

• Molecular cloning: The process of inserting DNA fragments into vectors (e.g., plasmids) for propagation and analysis.

Summary Table: DNA and RNA Comparison

Additional info:

• DNA is the hereditary material, as demonstrated by classic experiments (Avery, Hershey-Chase).

• DNA replication is semiconservative: each daughter molecule contains one parental and one new strand.

• Special DNA structures (triple helices, G-quadruplexes) may play roles in regulation and chromosome stability.