335

LECTURE NOTES BCH335 BIOCHEMICAL GENETICS AND BIOTECHNOLOGY By FRANCIS K ABROKWAH (PhD)

DNA

  • Definition: Deoxyribonucleic acid (DNA) is a polymer of deoxyribonucleotides. It is usually double-stranded and has a double-helix structure.

  • Location: Found in chromosomes, mitochondria, and chloroplasts.

  • Function: Acts as the genetic material in most organisms, carrying genetic information.

Structure of Nucleotides

  • Components:

    1. Pentose Sugar: Can be ribose (for RNA) or deoxyribose (for DNA).

    2. Phosphate Groups: Typically attached to the 5' position of the sugar.

    3. Nitrogenous Bases: 5 major bases found in cells:

    • Purines: Adenine (A), Guanine (G) (found in both DNA and RNA).

    • Pyrimidines: Thymine (T), Cytosine (C) (found in both DNA and RNA), Uracil (U) (found in RNA).

  • Nucleotides Composition:

    • 5-carbon sugar (ribose or deoxyribose) + phosphate group + nitrogenous base.

Nucleotide Structures of Purine and Pyrimidine

  • Purines: Double-ring structures (Adenine and Guanine).

  • Pyrimidines: Single six-sided ring structures (Cytosine, Thymine, and Uracil).

    • Basic Structures:

    • Adenine: C5H5N5

    • Guanine: C5H5N5O

    • Cytosine: C4H5N3O

    • Thymine: C5H6N2O2

    • Uracil: C4H4N2O2

Nucleosides

  • Definition: Nucleosides consist of a purine or pyrimidine base linked to a sugar (β-D-ribose or β-D-2-deoxyribose) via a covalent β-N-glycosidic bond.

  • Forming Nucleosides: A nucleoside is formed by joining a nitrogenous base to a sugar through a β-N-glycosidic bond, typically to N-1 of a pyrimidine or N-9 of a purine.

Phosphate Group

  • Charge: Negatively charged at neutral pH.

  • Role: Each nucleotide in nucleic acids contains one phosphate group.

Discovery of DNA Structure

  • Historical Events:

    • 1868: Friedrich Meischer identified DNA in the nucleus and named it 'Nuclein'.

    • 1953: James Watson and Francis Crick described the double helix model with contributions from Rosalind Franklin (X-ray diffraction), Maurice Wilkins, and Linus Pauling.

DNA Structure

  • Basic Components:

    1. Deoxyribose (pentose sugar)

    2. Nitrogenous Base (four types)

    3. Phosphate

  • Backbone: The structure consists of a sugar-phosphate backbone and nitrogenous bases.

Double Helix Structure

  • Characteristics: Two polynucleotide chains, anti-parallel polarity (5'-3' and 3'-5').

  • Base Pairing:

    • Adenine (A) forms 2 hydrogen bonds with Thymine (T).

    • Guanine (G) forms 3 hydrogen bonds with Cytosine (C).

  • Complementarity: Chargaff's Rule: %A = %T and %G = %C; summed should equal to 100%.

  • Hydrogen Bonds: Key to stabilizing the DNA structure.

Chargaff's Experimentation

  • Observations: Analyzed DNA base composition in various species and concluded percentages.

  • Chargaff's Rules:

    • Base pair contributions indicate A=T and G=C.

DNA Helix Physical Properties

  • Pitch: Approximately 3.4 nm; about 10 base pairs per complete turn.

  • Base Pair Distance: Each base pair is separated by approximately 0.34 nm.

  • Helical Structure Grooves: Major and minor grooves provide binding sites for proteins.

Forms of DNA

  1. B-DNA: Most common form, right-handed helix; under normal physiological conditions.

  2. A-DNA: Right-handed, observed under dehydrated conditions; has 11 base pairs per turn.

  3. Z-DNA: Left-handed, suggested presence during transcription regulation, zigzag pattern, and 12 base pairs per turn.

DNA Supercoiling

  • Definition: The over- or under-winding of DNA strands, important for DNA packaging in cells.

  • Structure: Chromosomes appear as supercoiled strands; essential for fitting DNA into cells.

Nucleosome Structure

  • Basic Packaging Unit: Nucleosomes consist of DNA wrapped around histone proteins.

  • Histones: Rich in lysine and arginine; assemble into octamers that associate with DNA to form chromatin.

Genome Characteristics

  • Genes: Segments of DNA coding for specific polypeptides or RNAs.

  • Genome: Entire DNA content of an organism.

  • C-Value Paradox: The amount of DNA does not necessarily correlate with organismal complexity.

Gene Expression Overview

  • Process: Gene expression involves two main stages - transcription and translation.

  • Central Dogma: Flow of genetic information: DNA → RNA → Protein.

DNA Sequencing and Applications

  • Purpose: To determine ordered sequences of nucleotides in DNA.

  • Methods:

    1. Maxam and Gilbert Sequencing: Based on chemical cleavage.

    2. Sanger Sequencing: Chain termination method using dideoxynucleotides.

Gene Cloning Overview

  • Definition: Involves isolating and copying segments of DNA (genes) to produce recombinant DNA.

  • Key Components: Vectors (plasmids), restriction enzymes, and ligases.

  • Process Steps: Insertion of DNA into a vector, transformation into host cells, and selection for successful clones.

Recombinant DNA Technology

  • Applications: Used in medical, agricultural, and environmental biotechnology to produce specific proteins and modify organisms genetically.

  • Purification & Verification: Techniques include gel electrophoresis and plasmid extraction methodologies.

Conclusion

  • These notes summarize critical concepts in Biochemical Genetics and Biotechnology, including the structure of DNA, genetic sequencing, and applications of recombinant DNA technology. Students are urged to consult original materials for in-depth study and understanding of the pivotal elements discussed in this area.