Vets Chapter 0 and 1

Chapter 0 & 1 Overview

DNA Composition

  • Deoxyribose Sugar: DNA includes a 5-carbon sugar known as deoxyribose, crucial for its structural integrity.

  • Nitrogenous Bases: Consists of four bases - Adenine (A), Thymine (T), Cytosine (C), and Guanine (G).

  • Phosphate Group: Each nucleotide contains a phosphate group with one phosphorus atom bonded to four oxygen atoms, contributing to DNA’s negative charge.

Properties of DNA

  • Solubility: DNA is soluble in water and can be stained using Ethidium Bromide, allowing visualization under UV light.

  • Denaturation: DNA can undergo denaturation (separation of strands) and renaturation (reforming double strands), which is essential for various laboratory techniques.

RNA Composition

  • Composed of Ribose Sugar, and has four nitrogenous bases: Adenine (A), Uracil (U), Cytosine (C), and Guanine (G).

  • Key Differences:

    • Ribose (in RNA) vs. Deoxyribose (in DNA) (the 2' carbon in DNA lacks an oxygen atom).

    • Strand Structure: DNA is double-stranded, while RNA is usually single-stranded and less stable.

    • Base Replacement: Thymine is replaced with Uracil in RNA.

    • Self-Replication: DNA has the ability to self-replicate.

Nucleotide Structure

5-Carbon Sugar

  • Each carbon is denoted by a prime nomenclature (e.g., 3' and 5').

  • The 3' and 5' positions are where nucleotides bond together to form a poly-nucleotide chain.

Phosphate Group

  • Structured with one phosphorus atom and four oxygen atoms, linking at the 5' carbon of the sugar.

  • The phosphate group carries a negative charge, influencing the structure and function of nucleic acids.

Nitrogenous Bases & Bonding

Purines and Pyrimidines

  • Purines: Characterized by two rings. Includes Adenine and Guanine. Mnemonic: "Pure As Gold".

  • Pyrimidines: Single-ringed bases include Cytosine, Uracil (in RNA), and Thymine (in DNA). Mnemonic: "CUT".

Base Pairing Rules

  • Adenine pairs with Thymine (DNA) or Uracil (RNA) via 2 Hydrogen Bonds.

  • Guanine pairs with Cytosine via 3 Hydrogen Bonds.

DNA Structure & Replication

Antiparallel Structure

  • DNA strands run in opposite directions (one is 3' to 5' and the other is 5' to 3'), which is essential for interactions between strands.

Semiconservative Replication Model

  • Each new double helix consists of one parental strand and one newly synthesized strand, highlighting the process of DNA replication leading to complementary base pairing.

Central Dogma of Molecular Biology

DNA Replication

Initiation:

  1. Occurs at the origin of replication (oriC).

  2. Helicase unwinds the strands, breaking hydrogen bonds between bases.

  3. Topoisomerase helps relieve the tension created by unwinding DNA.

  4. Single-stranded binding proteins stabilize the unwound strands.

  5. Primers, synthesized by RNA primase, bind to start replication.

Elongation:

  1. DNA polymerase attaches to RNA primers to add complementary nucleotides.

  2. The leading strand is synthesized continuously, while the lagging strand forms in segments called Okazaki fragments.

Termination:

  1. Exonucleases remove RNA primers and proofread the DNA.

  2. DNA ligase seals nicks between segments, forming a complete strand.

DNA Transcription

Initiation:

  1. Begins at promoter sites recognized by transcription factors.

Elongation:

  1. RNA polymerase utilizes the DNA template to synthesize RNA from 5' to 3'.

Termination:

  1. Transcription concludes at terminator sequences, leading to RNA strand release.

mRNA Processing

  • Capping and Tail Addition: A methylated cap is added to the 5' end, and a Poly-A tail is added to the 3' end to protect RNA integrity and facilitate transport from the nucleus.

  • Splicing: Introns (non-coding regions) are removed, and exons (coding sequences) are joined to produce mature mRNA.

RNA to Protein Translation

Initiation

  1. The ribosome assembles around the mRNA, beginning with the start codon AUG.

Elongation

  1. tRNA molecules bring amino acids corresponding to mRNA codons, forming a polypeptide chain, which is extended as the ribosome moves along the mRNA.

Termination

  1. Translation stops when a stop codon is encountered, leading to the release of the polypeptide and dissociation of the ribosome.

Protein Structure

  • Primary Structure: A simple polypeptide chain.

  • Secondary Structure: Folds in polypeptides, forming structures like alpha helices and beta pleated sheets.

  • Tertiary Structure: This structure emerges from interactions among R groups, enabling unique 3D shapes.

  • Quaternary Structure: Consists of multiple polypeptide chains interacting to form a functional protein.

Cloning and DNA Libraries

Cloning Process Overview

Step 1: Cleavage

  • Involves using restriction enzymes to digest DNA fragments for cloning.

Step 2: Ligating

  • DNA ligase is then added to anneal the vector and insert together.

Step 3: Transforming & Amplifying

  • The recombinant vector is introduced into bacteria, allowing replication and cloning of the DNA fragment.

Plasmids and Their Application

  • Plasmids: Circular DNA structures in bacteria, functional as vectors for inserting additional DNA sequences for cloning experiments.

  • Transformation Issues: Normal cells have negative charges, making plasmid uptake difficult, which can be facilitated by treatments that alter membrane charge.

Identifying Transformed Cells

  • Selection Markers: Use of resistant genes (such as Ampr) to identify successful transformations using antibiotic plates.

  • X-Gal Screening: Bacteria with an insert will remain white whereas those without the insert become blue due to enzymatic activity from the lacZ gene.

Types of DNA Libraries

Genomic Libraries

  • Collections of DNA fragments from an organism’s entire genome, including coding and non-coding regions.

cDNA Libraries

  • Comprised of only the expressed genes from an organism, generated by synthesizing cDNA from mRNA.

Comparison of Libraries

  • Genomic libraries retain all genomic information while cDNA only contains coding sequences, essential for understanding gene expression.

Constructing cDNA Libraries

  1. Isolate mRNA: Exploit the poly-A tail to separate mRNA from other RNA types.

  2. Reverse Transcription: Use reverse transcriptase to synthesize cDNA.

  3. Second Strand Synthesis: Create a double-stranded cDNA.

  4. Ligating into a Vector: Insert cDNA into appropriate vectors ensuring directional insertion using compatible restriction sites.

Summary

The successful execution of cloning and synthesis of cDNA libraries is pivotal for gene expression studies and understanding the functional genetics of organisms. This foundational knowledge underscores the intricate relationship between DNA, RNA, and protein synthesis, highlighting the central dogma of molecular biology.