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BIO205-CH9-rDNAtech

BIO205 - CH9 - Recombinant DNA Technology - Tortora - Rio Salado - AZ

QuestionAnswer
How can microorganisms & plants be used as "factories" to produce unnatural chemicals/products? By inserting genes into cells by recombinant DNA tech (rDNA) - genetic engineering
Another name for recombinant DNA (rDNA) technology. genetic engineering
vector A plasmid or virus used in genetic engineering to insert genes into a cell.
DNA vectors are often called __. gene-cloning vectors, or cloning vectors.
Site-directed mutagenesis. Techniques used to modify a gene in a specific location to produce the desired polypeptide.
Site-directed mutagenesis can be used to __. make a specific change in a gene.
restriction enzymes Special class of DNA - cutting enzymes that exist in many bacteria.
Restriction enzymes protect bacteria from __. a hydrolyzing phage DNA - protected because cell methylates (add methyl groups).
methylates Using restriction enzymes to add a methyl group to cytosines in DNA.
What role do restriction enzymes play? Recognizes & digests (cuts) only are particular sequence of nucleotide bases in DNA.
What is useful about sticky ends left by staggered cuts? They "stick" to complementary stretches of single-stranded DNA by base-pairing. Same enzymes used on 2 different sources of DNA will splice (recombine) in vitro.
Order of formation of rDNA molecule. (1) restriction enzyme cuts 2 DNA at its particular site, (2) sticky ends formed, (3) sticky ends bond w/hydrogen bonds, (4) DNA ligase covalently bonds backbones of DNA pieces to produce rDNA molecules.
Why are restriction enzymes used to make recombinant DNA? Because they produce staggered cuts that create "sticky ends" that can bond w/other DNA strands cut by same enzymes.
Most important property of a vector. self-replication
What does an ori (origin of replication) allow a plasmid to do? be self-replicating
Vectors serve as __. vehicles for the replication of desired DNA sequences.
Which size of vector is easier to manipulate? Smaller vectors, because larger DNA molecules are more fragile.
Most important property of a vector. self-replication
What does an ori (origin of replication) allow a plasmid to do? be self replicating
Vectors serve as __. vehicles for the replication of desired DNA sequences.
Which size of vector is easier to manipulate? Smaller vectors, because larger DNA molecules are more fragile.
What is special about shuttle vectors? They are plasmids that are capable of existing in several different species & can be used to move cloned DNA sequences among speces.
Viral DNA Vector that can usually accept much larger pieces of foreign DNA than plasmids can.
Name some viruses that are used as viral vectors. Retro viruses, adenoviruses, & herpes viruses.
Polymerase chain reaction (PCR) Technique by which small samples of DNA can be quickly amplified (increased quantities).
PCR can amplify __. small samples of DNA
primers Short pieces of nucleic acid added to help start reactions - complementary to ends of target DNA & hybridize fragments.
After each cycle of synthesis, DNA is __ to convert all the new DNA into a single strand. heated
What enzyme copies DNA in PCR? DNA polymerase
DNA polymerase for PCR comes from __. Thermus aquaticus - a thermophilic bacterium - allows for heating by a thermalcycler.
PCR __ be used to amplify an entire genome. cannot
In genetic engineering, a plasmid is inserted into a cell by __. transformation
transformation Procedure during which cells can take up DNA from environment - cells soaked in Ca chloride & made competent - heat added & then they take up DNA.
electroportation Using electric current to create micro pores in membrane - cells then absorb DNA through pores.
protoplasts Cell walls removed enzymatically to make bacteria vulnerable to experimentation.
Protoplast fusion Protoplasts in a solution will fuse & create hybrid cell - can merge species like plant & alge.
Microinjection inserting DNA into a cell w/glass micropipette
Foreign DNA will only survive if it __. is present on self-replicating vector or is incorporated into a chromosome by recombination.
gene library collection of clones containing different DNA fragments.
Genes are cloned into vectors using __. restriction enzymes
The enzyme reverse transcriptase is used to synthesize __. complementary DNA (cDNA)
cDNA is synthesized from __ template. an mRNA
A DNA copy of mRNA is produced by __. reverse transcriptase
cDNA DNA made in vitro from an mRNA template.
The __ method is most common method of obtaining eukaryote genes. cDNA
How does cDNA differ from synthetic DNA. Synthetic DNA is created mechanically by assembling nucleotides as if letters of a keyboard, while cDNA contains exons from a mRNA template.
Blue-white screening for clones If a bacterium received the original plasmid containing a piggy-backed gene, it will hydrolyze x-gal & produce a blue-colored compound.
Colony hybridization ID of a colony containing a desired gene by using DNA probe complementary to that gene.
DNA probe Short, labeled, single strand of DNA or RNA used to locate its complementary strand in a quantity of DNA.
DNA probes are labeled with? A radioactive element of fluorescent dye.
In blue-white screening, white contains? Foreign DNA
In blue-white screening, blue contains? no foreign DNA
Why is E. coli used in genetic modifications. They are grown easily & their genetics are known.
What is one disadvantage of using E. coli for genetic engineering. They are gram-negative & produce endotoxins.
Why use yeast cells in genetic engineering? They are eukaryotic & may carry plasmids.
gene silencing Defense against viruses & transposons.
RNA interference (RNAi) Target a particular gene - like a virus gene - and binds to mRNA & destroys it - silencing the expression of the gene.
One technique for genome sequencing is __. random shotgun sequencing.
Random shotgun sequencing Technique for determining the nucleotide sequence in an organism's genome.
bioinformatics The science of understanding the function of genes through computer-assisted analysis.
proteomics Science of determining all of the proteins expressed in a cell.
2 uses of Southern blotting Genetic screening & DNA fingerprinting.
Created by: Ladystorm
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