Nucleic Acids: Structure, Extraction, and Analysis

  • Structure of DNA and RNA

    • DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are polymers of nucleotides.
    • Nucleotide structure:
    • DNA: deoxyribose, thymine (T), cytosine (C), guanine (G), adenine (A).
    • RNA: ribose, uracil (U), cytosine (C), guanine (G), adenine (A).

  • Key Features of Nucleic Acids

    • DNA strands are anti-parallel and complementary:
    • A pairs with T (2 hydrogen bonds)
    • G pairs with C (3 hydrogen bonds)
    • Sugar-phosphate backbone formed by phosphodiester bonds.

  • Different Forms of DNA

    • A-DNA: Right-handed, occurs in dehydrated conditions, unstable.
    • B-DNA: Right-handed, common form under physiological conditions.
    • Z-DNA: Left-handed, may regulate gene expression and genetic recombination.

  • Nucleic Acid Structures

    • Cruciform: Important for gene expression.
    • Hairpin: Common in secondary structures of DNA and RNA.
    • Loop/Bubble: Formed due to unpaired nucleotides or enzyme action.

  • Supercoiling

    • Plasmid DNA exists in a supercoiled form.
    • Supercoiling is the most stable form.

  • Nucleic Acid Extraction

    • Release cell components: Lysis buffer with enzymes (e.g., Proteinase K).
    • Removing unwanted nucleic acids using DNase or RNase.
    • Purification methods:
    • Solvent extraction with phenol/chloroform.
    • Solid phase extraction using silica columns or magnetic beads.

  • Measuring Nucleic Acids

    • Spectrophotometry: Measures absorbance at 260 nm.
    • A260/A280 ratio indicates purity (1.8 for DNA, 2.0 for RNA).
    • Fluorimetry: More sensitive, uses fluorescent dyes to bind selectively to nucleic acids.
    • Gel electrophoresis: Separates nucleic acids by size under an electric field.

  • Denaturation & Renaturation

    • Denaturation: Separation of strands due to heat/pH.
    • Renaturation (annealing): Formation of complementary base pairing at lower temperatures.

  • Hybridisation Techniques

    • Southern Blot: Identifies specific DNA fragments using gel electrophoresis and hybridisation.
    • Microarray: Simultaneous detection of multiple sequences, quantifies presence based on fluorescence.

  • Important Notes

    • Understand structures of DNA and RNA.
    • Know extraction processes and comparison of methods for pDNA extraction.
    • Be familiar with spectrophotometry and fluorimetry for nucleic acid quality assessment.
    • Compare gel electrophoresis techniques and understand factors affecting nucleic acid migration.
    • Recognize applications of hybridisation methods in analyzing DNA.