DNA Technologies Review

CHAPTER 9: DNA Technologies

DNA Cloning

  • Definition: The process of creating identical copies of a piece of DNA (gene) or organism.
  • Types of cloning:
    • Organism cloning: Creation of identical copies of an organism.
    • DNA cloning: Focuses on isolating a specific gene from a source organism and amplifying it in a target organism.
  • Basic Steps of DNA Cloning:
    1. Cut the source DNA at the boundaries of the gene using restriction endonucleases.
    2. Select a suitable carrier DNA (vector) to insert the gene.
    3. Insert the gene into the vector using DNA ligase.
    4. Insert the recombinant vector into a host cell.
    5. Let the host cell produce multiple copies of the recombinant DNA.

Recombinant DNA

  • Definition: Artificially created DNA that combines sequences that do not normally occur together in nature.
  • Applications:
    • Molecular cloning of genes.
    • Over-expression of proteins.
    • Production of transgenic food and animals.

DNA Cloning Process

Step 1: Generate Recombinant Vector
  • Cloning Vector: Usually a plasmid that is cleaved with restriction endonuclease to prepare for ligation with the DNA fragment of interest.
  • Fragment Ligation: Fragments obtained from eukaryotic chromosomes are ligated to the prepared cloning vector using DNA ligase.
Step 2: Introduce DNA into Organism
  • Host Cell Transformation: Insert the recombinant vector into the host cell, which will propagate the transformed cell, producing many copies of recombinant DNA.

Cloning Vectors

  • Definition: DNA molecules used to carry foreign genetic material into another cell.
  • Types of Cloning Vectors:
    • Plasmids:
    • Circular DNA separate from the bacterial genomic DNA.
    • Autonomously replicating with origins of replication.
    • Can carry antibiotic resistance genes (e.g., Ampicillin, Tetracycline).
    • Can clone DNA segments up to 15,000 base pairs (bp).
    • Bacterial Artificial Chromosome (BAC):
    • Used in bacteria; can clone fragments up to 300,000 bp.
    • Yeast Artificial Chromosome (YAC):
    • Used in yeast for cloning larger fragments.

Restriction Endonucleases

  • Function: Enzymes that cleave DNA phosphodiester bonds at specific sequences.
  • Types of Cuts:
    • Staggered Cuts (Sticky Ends): Allow for easier ligation of pieces of DNA together.
    • Straight Cuts (Blunt Ends): Less flexible for recombination but still useful.
  • Applications: Widely utilized in molecular biology and genetic engineering for inserting genes into vectors.

DNA Ligase

  • Function: Enzyme that covalently joins two DNA fragments.
  • Role in Cloning: Ligates the DNA fragment of interest to the cloning vector, facilitating the formation of recombinant DNA.
  • Types: Human DNA ligase uses ATP whereas bacterial DNA ligase uses NAD.

Antibiotic Selection

  • Purpose: To select for bacteria that have taken up plasmids carrying antibiotic resistance genes.
  • Procedure:
    1. Cleave the plasmid vector at the antibiotic resistance element and ligate foreign DNA.
    2. Transform E. coli cells with the recombinant plasmid.
    3. Select transformed cells by plating on agar containing tetracycline and observing growth (those with ligations will not have ampicillin resistance).

Separation of DNA by Electrophoresis

  • Mechanism: Negatively charged DNA migrates towards the anode when placed in an electric field. The agarose gel hinders DNA mobility based on size and shape.
  • Application: Use in DNA analysis, purification, and studies of DNA-protein interactions.

Expression of Cloned Genes

  • Objective: To study the protein product of a gene after cloning.
  • Expression Vectors: Specialized plasmids that contain components necessary for gene expression, including:
    • Promoter and operator sequences.
    • Ribosome binding site.
    • Transcription termination sequences.

Site-Directed Mutagenesis

  • Purpose: Identify and explore the function of specific amino acids in proteins by mutating them.
  • Process:
    1. Change nucleotides in coding DNA using synthesized primers.
    2. Incorporate these changes into newly synthesized DNA.
    3. Sequence mutated plasmids to confirm the desired change is present.

Purification of Recombinant Genes

  • Process: Involves tagging recombinant proteins for easier purification using affinity resin techniques.
  • Example: A GST tag can bind to a resin, allowing for the purification of the protein of interest from a mixture.

Polymerase Chain Reaction (PCR)

  • Purpose: Amplify DNA sequences exponentially in vitro.
  • Components:
    • Target DNA.
    • Specific primers (short oligonucleotides complementary to the target).
    • Nucleotides (dATP, dCTP, dGTP, dTTP).
    • Thermostable DNA polymerase.
  • Process:
    1. Denature the DNA at 95°C.
    2. Anneal primers at 50-60°C.
    3. Extend primers at 75°C, repeating these steps for amplification.
  • Amplification Result: Over 20 cycles can amplify the target sequence by about 1,000,000 fold.

DNA Fingerprinting

  • Concept: Uses short tandem repeats (STR) that vary in number among individuals to identify genetic differences.
  • Method: Amplify specific regions using PCR and analyze fragment sizes for identification.
  • Database: 13 STR loci are used for forensic identification with high specificity (less than 1 in $10^{18}$ for misidentifications).

Adaptations to PCR

  • Reverse Transcriptase PCR (RT-PCR): Amplifies RNA by first converting it into cDNA.
  • Quantitative PCR (Q-PCR): Measures gene expression levels quantitatively.

Eukaryotic Gene Expression in Bacteria

  • Challenge: Eukaryotic genes contain introns that must be spliced out; bacteria lack splicing machinery.
  • Solution: Use complementary DNA (cDNA) synthesized from mRNA, which contains no introns for expression in bacteria.

Construction of cDNA

  • Procedure:
    1. Extract mRNA from eukaryotic cells, which have a poly-A tail for purification.
    2. Use reverse transcriptase to synthesize cDNA from mRNA.
    3. Convert the hybrid mRNA-DNA into double-stranded DNA, known as cDNA.

Visualization of Proteins

  • Methods:
    • GFP Tagging: Attaching green fluorescent protein to proteins of interest allows visual tracking using fluorescence microscopy.
    • Immunofluorescence: Tagging proteins with antibodies that are fluorescently labeled.

Identifying Protein-Protein Interactions

  • Tandem Affinity Purification (TAP): Enhances purification and identification of protein complexes by using dual purification steps.
  • Yeast Two-Hybrid System: Technique to identify physical interactions between proteins using transcriptional activation.

DNA Microarrays

  • Purpose: Allow for high-throughput screening of gene expression across thousands of genes simultaneously.
  • Method: mRNA/cDNA is tagged and hybridized to microarrays; analysis shows differential gene expression between samples.

New Generation Sequence Analysis

  • Techniques:
    • Pyrosequencing: Synthesizes DNA, generating light pulses that correlate with nucleotide additions.
    • Reversible Terminator Sequencing: Detected fluorescently labeled nucleotides are added in cycles to determine sequences.

The Human Genome Project

  • Goals: Sequence the entire human genome to identify genes associated with diseases and understand human genetic diversity.
  • Findings: Identified differences in haplotypes, genes involved in diseases, and ancestral migration paths based on genomic analysis.

Summary of Chapter 9

  • Key learned elements:
    • Techniques for creating recombinant DNA.
    • Methods of DNA cloning using bacterial systems.
    • Analytical techniques for DNA size and sequence.
    • Approaches for mutating and amplifying DNA in vitro.
    • Processes for expressing and purifying eukaryotic genes.
    • Techniques to measure gene expression levels effectively.