DNA Manipulation: Enzymes

• Enzymes manipulate DNA through synthesis, joining, and cutting.

Endonucleases

• Enzymes that cut DNA by breaking phosphodiester bonds between nucleotides.

Restriction Enzymes

• A type of endonuclease produced by bacteria, cuts DNA at specific restriction sites.
• Sticky ends: Staggered cuts with overhanging nucleotides, creating complementary single-stranded overhangs that attract each other.
• Blunt ends: Straight cuts resulting in no overhanging nucleotides, less favorable for DNA manipulation.

Ligases

• Enzymes that join nucleic acid fragments by creating phosphodiester bonds.
• Cannot create new DNA or hydrogen bonds between base pairs.

Polymerases

• Enzymes that join nucleotides to create new nucleic acids, and amplify DNA.
• Requires a primer, a short complementary strand of nucleic acid, to begin DNA strand synthesis (e.g., Taq polymerase in PCR).
• Polymerases synthesize new strands using a complementary strand, unlike ligases that connect short fragments.

CRISPR-Cas9 Function

• CRISPR-Cas9 consists of Cas9 endonuclease and sgRNA (single guide RNA).
• It is a quicker, cheaper, and more accurate way of editing an organism’s DNA.
• Cas9 can be programmed to cut at different points by modifying its sgRNA, unlike other endonucleases.
• Evolved in bacteria as an immune system against bacteriophages by cutting invading viral DNA and embedding it into CRISPR sequence.

Key Terms

• sgRNA/gRNA: Pre-modified RNA complementary to the DNA site of interest, directing Cas9 enzyme where to cut; programmed by scientists to cut at any known target gene.
• gRNA is a general term (immune), sgRNA is a specific type of gRNA (DNA manipulation).
• Cas9 endonuclease: Unzips double-stranded DNA and cuts the strands when a sequence complementary to the sgRNA is found.
• PAM sequence: A short DNA sequence providing the binding site for Cas9; essential for Cas9 to cut DNA.
• In bacteria, PAM protects against self-cutting, as bacterial DNA lacks this sequence.
• In gene editing, Cas-9 looks for this sequence to cut.

CRISPR-Cas9 Application for Gene Editing

• Identify the target gene sequence.
• Create sgRNA complementary to the target gene.
• Combine sgRNA with Cas9 and introduce into organism.
• sgRNA guides Cas9 to the target gene sequence.
• Cas9 cuts the target gene, altering the DNA sequence to switch off, repair, or alter the gene.

PCR (Polymerase Chain Reaction)

• Rapidly makes many copies (amplifies) of an original DNA sample.
• Used when there is insufficient DNA for testing, to amplify sections of DNA or entire polynucleotides.

Necessary Substances for PCR

• DNA sample: Serves as the template for amplification.
• DNA polymerase: Joins nucleotides to form new DNA strands.
• Nucleotides: Building blocks of DNA.
• Primers: Short single-stranded nucleic acids that act as the starting point for DNA polymerase.

PCR Steps

• Denaturing: DNA is heated to approximately 95°C to break hydrogen bonds and form single-stranded DNA.
• Annealing: The sample is cooled to 55°C to allow primers to bind to complementary sequences on the single-stranded DNA.
• Extending/Elongation: DNA is heated to 72°C (optimal temperature for Taq polymerase); Taq polymerase binds to primers and uses the original DNA strand as a template to synthesize a new DNA strand.
• Repeat: Steps 1-3 are repeated to create many copies of the original DNA strand.

Additional PCR Facts

• DNA strands are synthesized in opposite directions as Taq polymerase works in the 5’ to 3’ direction.
• DNA strands double each cycle, calculated as 2^n (n = number of cycles).

Taq Polymerase

• A heat-resistant DNA polymerase used in PCR to amplify DNA.
• High optimum temperature avoids denaturation during PCR cycles, allowing reuse.

Gel Electrophoresis

• A technique used to compare DNA samples by separating DNA fragments based on size.
• Samples are often amplified by PCR and cut with restriction enzymes first.

Steps of Gel Electrophoresis

• DNA samples and DNA ladder are loaded into wells at the negative electrode.
• An electric current is run through the agarose gel, causing DNA fragments to move towards the positive electrode.
• DNA fragments separate into bands, with smaller fragments moving faster than larger ones.
• The agarose gel is stained with fluorescent dye to visualize DNA bands under UV light.

Interpreting Gel Electrophoresis

• Thickness of the band indicates multiple fragments of that length.
• Plasmids with one restriction site form one strand; DNA with one restriction site forms two strands.
• DNA is negatively charged and moves towards the positive electrode.

Factors Influencing DNA Fragment Movements

• Voltage or power applied.
• Duration of the electrophoresis run.
• Viscosity of the agarose gel.
• Size of DNA fragments.

Properties of Genetic Code

• Universal: Nearly all organisms use the same codons for specific amino acids.
• Unambiguous: Each codon codes for only one specific amino acid.
• Degenerate/Redundant: Each amino acid may be coded by multiple codons, reducing the impact of mutations.
• Non-overlapping: Each triplet or codon is read independently.

Bacterial Transformation

• Many bacteria naturally take up free-floating DNA.
• Researchers use this process to make bacteria take up recombinant plasmids, turning them into protein-producing factories.

Key Terms

• Plasmid: Small circular DNA loop separate from the chromosome, found in bacteria.
• Plasmid vector: Circular DNA used to transport the gene of interest into bacteria.
• Restriction endonuclease: Cuts the gene of interest and the plasmid vector at specific restriction sites, creating complementary sticky ends.
• DNA ligase: Reattaches the DNA backbones.
• Antibiotic resistance gene: Selects for bacteria that have taken up a plasmid.
• Reporter gene: Selects for bacteria that have taken up the recombinant plasmid (e.g., GFP causes bacteria to glow green, LacZ glows blue).

Bacterial Transformation Steps

• Creating a recombinant plasmid: The gene of interest is removed using restriction enzymes, and the plasmid is cut with the same restriction enzymes to create complementary sticky ends.
• DNA ligase joins the gene of interest and plasmid, creating a recombinant plasmid.
• DNA polymerase can amplify the gene or fill in gaps if blunt ends are used.

Determining if Bacteria Uptake a Recombinant Plasmid

• Combine recombinant plasmids with bacterial cells and treat with heat or electricity to encourage uptake forming transformed bacteria.
• Select bacteria with a plasmid by growing them on an antibiotic plate; bacteria without a plasmid die.
• Determine which cells have a recombinant plasmid by checking if the reporter gene is expressed.
• Non-recombinant plasmids express the reporter gene; recombinant plasmids do not due to gene insertion.
• Transformed bacteria are cultured to transcribe and translate the new gene.

GMOs (Genetically Modified Organisms)

• Organisms with altered genetic material.
• Alterations include removing or inserting DNA, silencing genes, or replacing nucleotides.

TGOs (Transgenic Organisms)

• A type of GMO containing genetic material from another species.
• (e.g., Bt crops, golden rice).

Ethical Approaches

• Consequence-based: Maximizes positive outcomes while minimizing negative effects.
• Duty/rules-based: Follows a set of rules and responsibilities.
• Virtues-based: Driven by morals rather than rules or consequences.

Ethical Concepts

• Integrity: Honesty and accurate representation of facts.
• Beneficence: Maximizing benefits and minimizing harm.
• Non-maleficence: Acting to remove as much harm as possible.
• Justice: Fair distribution of resources and equal access to benefits.
• Respect: Considering the values of others, including their welfare, beliefs, freedom, and autonomy.