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UNLV / Biology / BIOL 251 / What are the properties of restriction enzymes?

What are the properties of restriction enzymes?

What are the properties of restriction enzymes?

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Exam 2 Study GuideIf you want to learn more check out Define neutrophil extracellular traps.

Lecture 5 Biotechnology - Modern applications of microbial genetics

Biotechnology - concept of using biological systems to produce productsWe also discuss several other topics like Define stereoisomers.

human manipulation of biological systems spans millennia, selective breeding

Recombinant DNA technology - manipulation of fragments of DNA containing

genes from one or more organisms, requires 3 componentsIf you want to learn more check out What are the different organ systems?

  • restriction enzymes endonuclease cleaving defined sequence
  • Gel electrophoresis procedure separate DNA fragments by size
  • DNA Ligase enzyme that joins different fragments of DNA

Don't forget about the age old question of What is a quantity that may change within the context of a mathematical problem or experiment?

Properties of restriction enzymes

Restriction site: specific sequence of DNA recognized and cutWe also discuss several other topics like What happened at the constitutional convention?
We also discuss several other topics like What order is a decomposition reaction?

Palindrome: sequence of DNA, often an inverted repeat.

  • sequence or complementary strands identical 5 to 3 direction

sticky ends: compatible cohesive end

blunt ends: each strand cut in same position, no overhang

Joining of DNA fragments

restriction digestion and gel electrophoresis isolated DN4 fragment

digestion with same restriction enzyme creates sticky ends

mixing of two DNA fragments allows hydrogen bond base pairing, transient

addition of DNA ligase allows formation of phosphodiester bonds

  • the two fragments are then permanently attached

modern cloning vectors: possess numerous restriction enzymes and more sophisticated selection schemes

  • Reporter gene: disrupted to detect ligation products
  • lacZ: turns on expression and detects activity lacZ activity
  • Multiple restriction sites engineered into lacZ gene
  • Insertion of DNA into any vector destroys lacZ activity
  • IPTG: controls gene product chemical “turns gene on”
  • Gene product has enzymatic activity, capable of detecting
  • X-gal can be broken down by gene product, dark blue
  • Blue white screening insertion of plasma containing DAN; presence of both IPTG + X-gal results in white colonies

generation of plasma genomic libraries

genome of organism of interest digested with restriction enzyme

inserted into plasmid digested with the same enzyme, involves ligation

  • each ligation event can produce different chromosomal fragments

transform bacteria with ligation mixture

must obtain enough transformants such that each gene is represented 1x

pool together transformants and harvest plasmid DNA

generation of phage libraries

genomic digest ligated into isolated phage genome

genome is transformed into bacteria generates plagues

  • each plaque can have a different genomic insert

Phage form plaques are pooled and propagated in appropriate bacterial strain

Generation of eukaryotic libraries

Propagates gene libraries into bacteria

  • bacteria lack machinery to remove introns
  • Results in production of DNA libraries to accomplish

mRNA is isolated and reverse transcriptase to produce DNA

competent bacteria's ability to take up DNA by growth in presence of it

  • Chemical competence: soak bacteria in metal salts to destabilize

The membrane, and heat shock to allow entry of DNA into cell

  • Electro competence: clean cells thoroughly with 10% glycerol

apply short burst of electrical current to destabilize membrane for DNA

cellulase /zymolyase: digestive enzymes used remove cell walls if necessary

  • spheroplast: a protoplast formed after breakdown of cell wall
  • removal of cell wall allows effective use of electroporation

gene gun: penetration of the cell using tungsten particles and helium gas

microinjection: insertion or DNA into nucleus through drawn glass pipette

Ti plasmid: tumor inducing plasmid

  • Can be used to form a recombinant plant through inserting the

gene of interest into me Ti plasmid, and then used to infect

the plan

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