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109 Cards in this Set
- Front
- Back
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Biotechnology
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using MOS to produce a desirable product
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To change the trait of an organism by introducing a foreign gene so that the organisms displays that gene as well as the valuable characteristics
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recombinant DNA
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Recombinant DNA
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to change the trait of an organism by introducing a foreign gene so that the organism displays the valuable characteristics of the new gene
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Genetic Engineering
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to manipulate genetic material to alter the characteristic of an organisms in a desired way
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What are the goals of genetic engineering?
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eliminate an undesirable trait from an organism and/or introduce a desirable trait to an organism
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What is making a cell porous so that it can take in DNA
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transformation
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What are physical or chemical agents that promote mutation
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mutagens
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What is RNA dependent DNA polymerase
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Reverse Transcriptase
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What has reverse transcriptase?
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only viruses
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What can reverse transcriptase do?
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if you have the enzyme, you can use RNA to make DNA
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What is making RNA from DNA
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transcription
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What is DNA made from RNA by some viruses
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complementary DNA
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What makes complementary DNA
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reverse transcriptase
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What is reverse transcriptase?
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RNA dependent DNA polymerase
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What occurs in eukaryotic transcription/translation?
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first you must copy the whole thing and get premRNA then it must be processed
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Is premRNA useful?
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no, it must be processed to be used
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Can you put the eukaryotic DNA into E. Coli?
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no, it must have the introns removed first, because E. Coli will not know what to do with the introns
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What is the coding part of eukaryotic DNA?
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exon
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What is the noncoding part of eukaryotic DNA?
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introns
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What is the waste nucleotides that are not used in eukaryotic DNA?
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introns
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What must be done to premRNA to put it into a prokayotie?
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it must be sliced to remove the introns and make mRNA
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What is inserted into a prokaryotic from a eukaryote?
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cDNA with no introns
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What is a desirable gene isolated from the donor organism?
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mRNA
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What is mRNA used for?
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a template to synthesize a complementary DNA
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What is the ss cDNA used for?
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to make a ds cDNA
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What is the ds cDNA?
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a copy of the desirable gene without introns that can be inserted into a prokaryotic recipient cells DNA directly
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What are introns
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noncoding part of eukaryotic DNA
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What are exons
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coding part of eukaryotic DNA
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What is hte major difference between prokarytoic and eukaryotic transcription?
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eukaryotic has pmRNA to mRNA
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Could a prokaryotic cell process a eukaryotic gene?
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no, it doesn't know how to cut out it's introns
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What is the major advantage to insert cDNA into a prokaryotic cell?
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cDNA has no introns
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WHat can a nucleic acid synthesizer do?
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can synthesize short pieces (up to 100 bases) of DNA or RNA by a DNA synthesizer and links them together
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What must you know to work a nucleic acid synthesizer?
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the sequence of the nucleotide
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What is PCR?
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polymerase chain reaction
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What does PCR do?
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replicate DNA in vitro
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What is the difference between replicating DNA in the cell and in vitro with the PCR?
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you can use DNAprimer in vitro instead of RNA primer and it is more stable
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What do you need for a PCR?
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DNA template, DNA primer, DNA polymerase (thermal stable), dATP, dTTP, dGTP, dCTP (building blocks of DNA, and thermocycler
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What are the building blocks of DNA?
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dATP, dTTP, dCTP, dGTP
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What does DNA polymerase add to?
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3' end
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What is a thermocycler?
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a mini incubator that changes temperature
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What are the 3 steps of PCR
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denaturation, priming, extension
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What occurs in the denaturation step of PCR?
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94 degrees C and the ds DNA splits into two ss DNA
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Why must the DNA polymerase in PCR be thermal stable?
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so that it can handle the 94 degrees C that occurs in denaturation as well as the other shifts in temperature
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What occurs in the priming step of PCR?
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65 degrees C and DNA primers bind to template
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What occurs in extension step of PCR?
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72 degrees C and DNA polymerase replicates DNA on both templates
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Can the primer be the same for both strands of DNA in PCR?
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no, if the primer doesn't match, it won't bind
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About how many cycles of PCR do you need to make 1 billion copies of identical DNA?
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about 30 cycles
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What produces restriction enzyme?
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bacteria in order to digest foreign DNA
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What is produced by bacteria to destroy/digest foreign DNA?
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restirction enzyme
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How do bacteria protect some of their own DNA?
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methylation of some nucleotides
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What cuts DNA at a specific sequence?
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restriction enzyme
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What is the site where a restriction enzyme cuts?
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a specific site called restriction site
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What are teh 2 kinds of restriction enzyme cuts?
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staggered and blunt
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What cut has a sticky end?
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staggered
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What is a small segment of DNA that can carry the desirable gene into the recipient cell?
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vector
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What are the important properties of vectors?
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small, can be adopted by the recipient, contains a genetic marker gene, provides required genetic elements for gene expression
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What can be introduced into host by transformation?
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plasmid
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What is a viral genome?
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some viruses can insert their DNA into host chromosomal DNA
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What can insert itself into host genome?
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transposon
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What sticks the sticky ends of plasmid vector together?
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liGASE
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What can be introduced to host cell?
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recombinant plasmid
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What is a collection of cloned recipient cells which contain different DNA fragments from the donor cell
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gene library
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WHat are the parts to peparing a target gene for DNA recombination
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cDNA, DNA syntehsizer, obtain the gene from gene library, multiply gene in PCR
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What are the steps in making recombinant DNA
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prepare target gene, select a host to express the gene, selct a vector, join the target gene into the vector's DNA, introduce the recombinant vector DNA into the host cell, identifying and selecting a clone of recombinant cell, grow the host cell that successfully receives the target gene, and harvest the useful product from the culture and purify the product as needed
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What are the vectors to select from for gene recombination?
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plasmid, virus, transposon
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How do you join the target gene into the vector's DNA?
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use specific enzyme to cut and use ligase to join the ends
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What are some difficulties in selecting a host?
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G- keeps the product in cytoplasm, but G+ secretes to environment
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HOw can you introduce the recombinant vector DNA into the host cell?
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transformation, transduction, protoplast fusion, microinjection, gene gun
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What facilities transformation?
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electroporation
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What does electroporation do?
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uses electrical current to treat recipient cells and to produce holes in teh cell membrane to make the cell competent for transformation
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What is transfection?
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allow virus to infect and bring DNA in: animal to animal or plant to plant
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difference between transduction and transfection
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transduction is bacteria phage: transfection is animal to animal or plant to plant
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What must you do with electroporation?
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control the dose so you don't kill the cell
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What makes protoplast?
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removing the cell wall
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What is protoplast fusion?
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protoplasts are treated with chemicals and the two protoplasts fuse into one hybrid cell
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What is microinjection
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DNA is inserted into a target cell using a micropipette: a lot of practice
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What is a gene gun?
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takes DNA coated beads and propels them into target cells
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What are 4 things that can happen with DNA recombination?
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the recipient cell gets... A) nothing, B) plasmid but no foreign DNA, C) recombinant plasmid with foreign DNA, or D) foreignt DNA without plasmid vector
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WHat does a probe do?
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tell the degree of hybridization
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What does it mean if theres some hybridization?
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organisms related
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Complete hybridization
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A and B identical, same DNA
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No hybridization
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A and B not the same
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What is a nucleic acid probe
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a short piece of ss DNA labeled with a radioisotope or other reported used to hybridize and detect its complementary ss DNA
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a short piece of ss DNA labeled with a radioisotope or other reported used to hybridize and detect its complementary ss DNA
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nucleic acid probe
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What is the point of nucleic acid probe
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to find the desirable gene
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What is electrophoresis?
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to separate DNA molecules
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What moves faster on electroporesis
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negative charaged and smaller molecules
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Electroporesis: which moves: + or -?
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negative charged migrate toward the + end
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Which move more rapidly: small or large?
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small
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What do you stain DNA bands with on the electrophoresis gel
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ehtidium bromide
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How do you locate target DNA with southern blot technigue
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DNA bands in gel transferred to nitrocellulose membrane and DNA probed used to detect target DNA on membrane
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What are some applications of recombinant DNA?
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crops, treat genetic disorder, gene therapy, vaccine, improving traits of plants and animals for agriculture, transgenic animals
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What is replacing defective genes with normal genes?
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gene therapy
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What is artificially acuiring bacteria cell to make antibodies
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vaccine
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What is a subunit vaccine
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don't use real virus, just virus structure because antibodies just need to bind to site/ very expensive
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What is combine vaccine
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stimulates to produce antibodies against multiple disease
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How can we improve plants and animals with DNA recombination?
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improve plants resistnace to herbicide, salt, freezing, pests, nutritional value
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What are transgenic animals
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using animals to make human products
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What are transplant of organs made by animals that are similar to humans?
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xenotransplant
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What are some other molecular techniques?
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dna fingerprinting, nucleotides sequencing, DNA microarrays
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What is DNA fingerprinting?
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isolate DNA from a sample, use PCR to multiply the DNAs, digest the DNA into pieces with a restriction enzyme, use gel electrophoresis to separate teh DNA pieces by size and make DNA patterns
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What will occur with DNA samples from the same source?
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should yield same response
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What is used to detect multiple target DNA or RNA in a smaple by ss DNA probes immobilized on a solid surface (such as a glass slide)?
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microarry (DNA chip)
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What does microarray do?
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glass side, silicon chip or nylon membrane that contains multiple ss DNA probes on surface and ss DNA strands from a smaple are labeled with fourescent dye and added to the DNA chip
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What can detect multiple genes in one sample?
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microarray
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What happens to those target DNA pieces complementary to DNa probes on the microarray?
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they will bind to the DNA chip and display the color of the flourescent dye
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What are applications of microarray?
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monitor gene expression (find out what type of mRNA are present in a cell) (mRNA --> cDNA) (add flourescent labeled cDNA to microarray), diagnosis of infection, identifying organisms in an environment sample
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What is genomics?
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DNA sequencing
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HOw does genomics work?
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sythesis of cDNA with flourescent dye labeled nucleotide and the DNA seqeuencer determines the sequence of color emitted by the DNA sample
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