Chapter 18 - Genetic Engineering Flashcards
Genetic Engineering
Deliberate manipulation of DNA, using techniques in the laboratory to alter genes in organisms.
Gene Cloning
Process by which a gene of interest can be replicated many times over.
Step 1 of Gene Cloning
Isolate DNA of interest. ie Glowing gene found in jelly fish, isolate the sequence responsible for this.
Cloning Vector
Typically a plasmid or virus, capable of independent replication that will stably carry the target DnA from one location to another
Step 2 of Gene Cloning
Cut the DNA with restriction endonucleases
endonucleases
Enzymes that recognize short sequences of DNA that are 4-8 bp long. They’re widespread in both bacteria and archaea.
How to endonucleases read DNA?
Palindromic (meaning same on each DNA strand in the 5’ to 3’ direction)
How do endonucleases cut DNA?
Some cut straight, many make staggered cuts, producing very short regions of single-stranded DNA.
sticky ends
Short region of single-stranded DNA on each strand after being cut by endonucleases. Invaluable in molecule cloning since unpaired bases will recombine with any DNA having the complementary base sequence.
Step 3 of Gene Cloning
Combine target and vector DNA - following cleavage of both types of DNA, the two types are then combined together with DNA ligase. Results in recombinant DNA.
DNA Ligase
Enzyme that repairs the covalent bonds on sugar-phosphate backbone of DNA.
Recombinant DNA
DNA molecules that contain the DNA from two or more sources, also known as chimeras
Step 4 of Gene Cloning
Introduction of recombined molecule into host cell.
Bacteria reintroduction
transformation is often easiest method, using competent cells to pick up the recombinant DNA.
electroporation
Also can be used for reintroduction, where cells are exposed to brief pulse of high-voltage electricity causing the membrane to become temporarily permeable to DNA passage
genomic library
Combination of both cells with new DNA and cells with the old DNA 0 must be screened to select appropriate clone
shotgun cloning
If random fragments of DNA were originally used, can result in thousands of tens of thousands of clones to be screened
Agrobacterium Tumefaciens
plant pathogen that causes tumor formation called crown gall disease. Contains plasmid known as Ti plasmid, which inserts bacterial DNA into host plan genome. Utilized by scientists to do genetic engineering of plants.
TI
Tumor Inducing
Gene Gun
Small metal particles coated with recombinant DNA, which as blasted at plan or animal tissue at high velocity. If DNA is transformed or taken up by cell’s DNA< the genes are expressed.
Viral Vectors
Virulence genes from a virus can be removed and foreign DNA inserted, allowing the virus capsid to be used as a mechanism for shuttling genetic material into a plant or animal cell.
Gel Electrophoresis
Technique commonly used to separate nucleic acid fragments based on size. Porous gel made of agarose with nucleic acid samples placed into wells. Electric current applied. Smallest particles will move fastest. Larger particles will move slower.
Polymerase Chain Reaction (PCR)
Method used to copy or amplify DNA in vitro. Can yield billionfold copies of a single gene. Template DnA is mixed with ingredients (primers, nucleotides, polymerase. Steps include denaturing DNA, drop temp to allow primers to anneal, and then reheat to allow DNA polymerase to extend.
Antisense RNA
ssRNA, complementary to mRNA that will code for a protein. Made as a way to control target genes. a way to prevent diseases that are caused by production of a particular protein.
