Waksman Vets Slideshows

DNA & RNA Composition

  • DNA Composition

    • Sugar: Deoxyribose

    • Bases: Adenine (A), Thymine (T), Cytosine (C), Guanine (G)

    • Phosphate Group

    • Properties:

      • Soluble in water

      • Stainable by Ethidium Bromide

      • Reflects UV light

      • Has a negative charge

      • Can be denatured and renatured

  • RNA Composition

    • Sugar: Ribose

    • Bases: Adenine (A), Uracil (U) instead of Thymine (T), Cytosine (C), Guanine (G)

    • Phosphate Group

  • Key Differences:

    • Ribose vs Deoxyribose: The 2’ in DNA lacks an oxygen

    • DNA is double-stranded; RNA is usually unstable and single-stranded

    • Thymine in DNA is replaced by Uracil in RNA

    • DNA is self-replicating

Structure of Nucleotides

  • 5-Carbon Sugar:

    • Each prime (') indicates nomenclature (3' and 5' are reactive groups)

    • Phosphate linked at the 5' carbon

    • Can have up to 3 phosphate groups (monophosphate, diphosphate, triphosphate)

  • Phosphate group:

    • Contains one phosphorus and four oxygens

    • Has a negative charge

Nitrogenous Bases

  • Purines:

    • Structure: 2 rings

    • Examples: Adenine (A) and Guanine (G)

    • Mnemonic: "Pure As Gold"

  • Pyrimidines:

    • Structure: 1 ring

    • Examples: Cytosine (C), Uracil (U), and Thymine (T)

    • Mnemonic: "CUT"

Base Pairing Rules

  • Adenine pairs with Thymine (DNA) or Uracil (RNA) via two hydrogen bonds

  • Guanine pairs with Cytosine via three hydrogen bonds

DNA Structure and Replication

  • Anti-Parallel Structure

    • One strand runs 5' to 3', the complement runs 3' to 5'

    • 3' end: free hydroxyl group

    • 5' end: free phosphate group

  • Semiconservative Model

    • New DNA strand synthesized in a 5' to 3' direction

    • Central Dogma: DNA -> RNA -> Proteins

  • DNA Replication Processes:

    • Initiation:

      • Starts at oriC (A and T-rich regions)

      • Helicase unwinds DNA, breaking hydrogen bonds

      • Topoisomerase prevents supercoiling

      • Single-stranded binding proteins stabilize unwound DNA

      • RNA primers are synthesized by primase

    • Elongation:

      • DNA polymerase adds complementary nucleotides

      • Leading strand is continuous (3' to 5'), lagging strand in fragments (Okazaki fragments)

    • Termination:

      • Exonucleases remove primers; DNA ligase seals fragments

RNA Transcription and Processing

  • Initiation of Transcription:

    • Begins at promoters with TATA proteins

  • Elongation:

    • RNA polymerase uses DNA template to synthesize RNA from 5’ to 3’

  • Termination:

    • Terminator sequences signal RNA polymerase to stop

  • mRNA Processing:

    • Addition of 5' cap and polyA tail

    • Splicing removes introns, joining exons to produce mature mRNA

Central Dogma: RNA to Protein

  • Initiation:

    • Ribosome assembles on mRNA; first tRNA brings methionine

  • Elongation:

    • tRNA matching codons brings amino acids to form polypeptide

  • Termination:

    • Stop codons signal release of polypeptide from tRNA

Protein Structure

  • Levels of Structure:

    1. Primary: sequence of amino acids

    2. Secondary: folding into alpha helices and beta sheets

    3. Tertiary: 3D structure formed from interactions between side chains

    4. Quaternary: multiple polypeptide chains form a functional protein

Cloning Process Overview

  • Cloning DNA Fragments:

    • Insert DNA into vectors using restriction enzymes

    • Transform into bacteria for amplification

    • Purify plasmids using miniprep method

  • Key Steps:

    • Digestion of DNA with restriction enzymes

    • Ligation to join DNA fragments

    • Transformation of plasmids into bacteria for growth

DNA Libraries

  • Types of DNA Libraries:

    • Genomic Libraries: include all genomic DNA fragments

    • cDNA Libraries: contain only expressed genes (complementary to mRNA)

  • Purpose:

    • Facilitate analysis of specific genes of interest

  • Construction of cDNA Libraries:

    1. Isolate mRNA

    2. Purify, reverse transcribe, and synthesize cDNA

    3. Ligate into vectors for transformation

PCR (Polymerase Chain Reaction)

  • Purpose: Amplify a specific DNA segment

  • Components:

    • Template DNA, primers, Taq polymerase, dNTPs

  • Process:

    • Denaturation, annealing, and elongation cycles result in millions of copies of DNA

Gel Electrophoresis

  • Use: Measure size of DNA fragments

  • Process:

    • DNA is loaded into a gel matrix and subjected to an electric field

    • Smaller fragments move faster

Restriction Enzymes

  • Function: Cut DNA at specific sequences

  • Use in Cloning: Allow for precise insertion of DNA fragments into vectors

  • Methylation: Protects host DNA from being cleaved

Sanger Sequencing

  • Purpose: Determine nucleic acid sequences

  • Process: Incorporates dideoxynucleotides to terminate extension, producing fragments of varying lengths that can be analyzed using electrophoresis

Next-Generation Sequencing (NGS)

  • Comparison to Sanger:

    • More efficient, can sequence millions of fragments simultaneously

Analysis Techniques

  • BLAST: To determine evolutionary relationships between sequences

  • Open Reading Frames (ORF): Identify potential coding sequences in nucleotides

Common Issues in Sequencing

  • Problems: Bad primers, poor template, or low-quality reagents can lead to unreadable sequences.

DNA Structure and Replication

Double-Stranded DNA (DSAP) Processes:

  • Anti-Parallel Structure: DNA consists of two strands that run in opposite directions, which is critical for replication and transcription processes.

    • The one strand runs from 5' to 3', while the complementary strand runs from 3' to 5'. This orientation affects how enzymes interact with the DNA during replication.

  • Semiconservative Replication: A fundamental characteristic of DNA replication where each new double helix contains one original strand and one newly synthesized strand. This mechanism preserves half of the original DNA molecule in each daughter cell, ensuring genetic fidelity.

Replication Stages:

  1. Initiation:

    • Replication Origin: The process begins at specific locations called origins of replication (oriC in prokaryotes). These regions have a high content of adenine (A) and thymine (T), which have fewer hydrogen bonds and are easier to separate.

    • Enzymatic Action: The enzyme helicase unwinds the double-stranded DNA by breaking hydrogen bonds between complementary bases, leading to the formation of replication forks.

    • Topoisomerase Function: Topoisomerases alleviate torsional strain that occurs ahead of the replication fork, preventing supercoiling by cutting and rejoining the DNA strands.

    • Stabilization: Single-stranded binding proteins (SSBs) bind to the strands to maintain separation and stabilize unwound regions of the DNA, preventing them from re-annealing.

    • RNA Priming: Primase synthesizes short RNA primers that provide the starting point for DNA synthesis by DNA polymerase. These primers are necessary because DNA polymerases can only add nucleotides to an existing chain.

  2. Elongation:

    • DNA Polymerase Activity: DNA polymerase III (in prokaryotes) or the corresponding enzyme in eukaryotes extends the new DNA strand by adding nucleotides complementary to the template strand in the 5' to 3' direction. The leading strand is synthesized continuously, while the lagging strand is synthesized as short fragments (Okazaki fragments) due to its opposite orientation.

    • Adjunct Enzymes: DNA polymerase I replaces the RNA primers with DNA nucleotides, sealing the gaps left by primers. Additionally, clamp loader complexes (like the sliding clamp) enhance the processivity of DNA polymerase, allowing it to remain attached to the template strand during replication.

  3. Termination:

    • Primer Removal: Exonucleases remove RNA primers after DNA synthesis, creating gaps in the newly formed DNA strands.

    • Ligation: DNA ligase enzyme fills in the gaps by catalyzing the formation of phosphodiester bonds between adjacent nucleotides, thus sealing the Okazaki fragments on the lagging strand.

    • Daughter Strand Formation: At the completion of these processes, two identical daughter DNA molecules are formed, each containing one original strand and one newly synthesized strand, ready for cell division.

Expanded DSAP Analysis Questions

  1. What is the structure of double-stranded DNA?

    • Anti-parallel Orientation: The strands run in opposite directions; one is oriented 5' to 3' while the other runs 3' to 5'. This is essential for the binding of complementary nucleotides and the action of enzymes during replication and transcription.

    • Base Pairing: Adenine (A) pairs with Thymine (T) via two hydrogen bonds, and Guanine (G) pairs with Cytosine (C) through three hydrogen bonds. This specificity ensures accurate DNA replication and integrity of genetic information.

    • Double Helix Formation: The strands twist around each other to form the characteristic double helix structure, stabilized by hydrogen bonding between bases and the hydrophobic interactions of the nucleotide bases.

  2. How does DNA replicate semiconservatively?

    • Semiconservative Replication: Each new double-helix consists of one old (template) strand and one newly synthesized strand. This mechanism allows for preservation of half of the original DNA in each daughter cell, which is critical for genetic fidelity during cell division.

    • Replication Forks: As DNA unwinds, replication forks are formed on either side, where the DNA is actively being synthesized.

  3. What are the key enzymes involved in DNA replication and their functions?

    • Helicase: Unwinds the double-stranded DNA at the replication fork by breaking the hydrogen bonds between complementary bases, providing single strands for replication.

    • Topoisomerase: Mitigates the supercoiling that occurs ahead of the replication fork by making temporary cuts in the DNA.

    • Single-stranded Binding Proteins: Stabilize the unwound strands and prevent them from re-annealing before replication occurs.

    • Primase: Synthesizes short RNA primers required for DNA polymerase to initiate synthesis, as it can only add nucleotides to an existing chain.

    • DNA Polymerase: Synthesizes new DNA strands by adding complementary nucleotides to the template strand in a 5' to 3' direction. Different types (e.g., DNA polymerase III in prokaryotes, DNA polymerase α, δ, and ε in eukaryotes) play roles in different replication processes.

    • DNA Ligase: Joins Okazaki fragments on the lagging strand by catalyzing the formation of phosphodiester bonds, sealing nicks in the sugar-phosphate backbone.

  4. What are common issues faced during DNA replication?

    • Incorrect Base Pairing: Misincorporation of nucleotides can lead to mutations if not corrected.

    • Template Issues: Damaged or low-quality DNA templates can result in an inability for DNA polymerase to replicate efficiently.

    • Low-Quality Reagents: Use of poor-quality nucleotides or enzymes can lead to incomplete or erroneous DNA synthesis.

    • Replication Stress: Conditions such as tension in the DNA due to tight winding or external factors can inhibit normal replication processes, leading to stalled replication forks.

By understanding these aspects of double-stranded DNA and its replication processes, students can appreciate the complexities of genetic material maintenance and expression.