Genetic Engineering: DNA Purification and Cloning Enzymes

• Genetic engineering involves DNA purification and manipulation.

DNA Purification

• Different types of DNA include Total DNA, Plasmid DNA, and Bacteriophage DNA.
• Chromosomal DNA is often separated from plasmid/phage DNA.
• Column chromatography can be used to purify DNA molecules.
• DNA can be purified with silica particles.

Plasmid Extraction

• Separation is based on size and conformational differences.
• Alkaline denaturation is a process used in plasmid extraction.
• Cesium chloride (CsCl) density gradient centrifugation separates DNA, RNA, and proteins based on density.
• Ethidium bromide (EtBr) can be extracted with n-butanol.
• CsCl is removed by dialysis.
• Phage suspensions can be purified through centrifugation.
• Phages can be precipitated with polyethylene glycol (PEG).

Cloning Enzymes

• Enzymes are used in DNA manipulation.
• Ligases join nucleic acid molecules.
• Polymerases create copies of molecules; examples include Pol I, Taq, and RT.
• Nucleases cut, shorten, or degrade nucleic acid molecules.
• Modifying enzymes are also utilized.
• DNA ligase links DNA strands with double-strand breaks and its applications include DNA replication and repair.
• DNA ligase use occurs in genetic recombination experiments for molecular biology laboratories.
• Exonucleases can remove nucleotides from both strands or only from the 3' terminus of DNA.
• Endonucleases include S1 nuclease, DNase I, and restriction endonucleases.

Restriction and Modification Enzymes

• Restriction enzymes protect cells from foreign DNA by destroying it and should protect cells own DNA.
• Restriction enzymes recognize specific DNA sequences and cut DNA at those sites.
• These are widespread among prokaryotes but rare in eukaryotes.
• Restriction enzymes protect prokaryotes from foreign DNA like viral genomes.
• They are essential for in vitro DNA manipulation.
• Type II restriction enzymes cleave DNA within the sequence recognized, useful for specific DNA manipulation.
• Restriction enzymes recognize inverted repeat sequences, or palindromes, typically 4-8 base pairs long, EcoRI recognizes a 6-base-pair sequence.
• Enzymes result in sticky or blunt ends.
• Modification enzymes protect a cell's DNA from restriction enzymes.
• They chemically modify nucleotides in the restriction recognition sequence, usually through methylation.
• Restriction endonucleases cannot bind to recognition sequences when sequences are methylated.
• A restriction digest involves adding a buffer, adding an enzyme, incubation, and then the addition of phenol or EDTA, or heat.

Agarose Electrophoresis

• Load samples in the electrophoresis device.
• Electrical current carries negatively-charged DNA through the gel towards the positive electrode.
• Agarose gel sieves DNA fragments based on size, with smaller fragments moving farther than large fragments.

Modification Enzymes

• Alkaline phosphatase
• Polynucleotide kinase
• Terminal deoxynucleotidyl transferase

Ligation

• Ligation is a step in DNA cloning and genetic engineering.
• Ligation involves the use of an enzyme called DNA ligase to join two DNA fragments together to create a single, continuous molecule of DNA.
• Ligation of blunt ends and sticky ends are two methods.
• Foreign DNA and a vector are cut with restriction enzymes, then DNA ligase forms recombinant molecules.

Inserting DNA into a Host Cell

• Transformation involves treatment to make competent cells for accepting foreign DNA, such as using CaCl2.
• Electroporation uses electrical current to create pores in membranes.
• Protoplast fusion is used for yeast, plants, and algal cells.
• Other methods include microinjection and gene gun.
• Transformation transfers exogenous DNA into cells.
• Methods includes a chemical method using CaCl2 and heat shock.
• Electroporation is another method with a short pulse of electric charge.
• Chemical transformation involves a calcium chloride solution, chilling cells on ice, heat shock, and then chilling on ice again.
• Electroporation involves sterile water in the process and electroporation instead of heat shock.

Transformation Results

• Bacterial cells can contain a plasmid with an insert, a plasmid without an insert, or no plasmid at all.
• Characteristics are that cells with:
• Plasmid with insert are ampicillin-resistant and have nonfunctional LacZ, create a white colony on LB+X-gal+amp
• cells with Plasmid w/o insert are ampicillin resistant and have functional LacZ, create a blue colony on LB+X-gal+amp
• cells with No plasmid have No ampicillin resistance and No LacZ gene, cause no growth on Ampicillin
• Recovery period allows cells to regain strength, facilitates expression of the antibiotic resistance gene on the plasmid, add sterile Luria Broth to both tubes, and use water at 37°C for 5 min.

Explore DNA purification techniques for different DNA types like Total DNA, Plasmid DNA, and Bacteriophage DNA. Learn about plasmid extraction methods such as alkaline denaturation and cesium chloride density gradient centrifugation. Understand the role of cloning enzymes like ligases, polymerases, and nucleases in DNA manipulation.