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30 Cards in this Set
- Front
- Back
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What is recombinant DNA technology? |
It is the strategy to capture specific DNA like: -a gene of interest -a gene that encodes a protein -DNA that has polymorphisms -A fragment of DNA that codes for the phenotype |
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What is the source of DNA used for cloning? |
-Usually genomic DNA (coding DNA) -also called cDNA |
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What is genomic DNA? |
-Genomic DNA is the DNA inside a eukaryote cell -mRNA (cDNA) codes for phenotype -DNA = 100% of genome -cDNA = 10% of genome |
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What are restriction enzymes (restriction endonucleases)? |
-They are the molecular scissors -Their function is to cut DNA |
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How do restriction enzymes discern between bacterial DNA and foreign DNA? |
-Restriction enzymes target foreign DNA because they are not methylated -Restriction enzymes can only bind to certain sites. If these sites are methylated, the enzyme cannot bind there. |
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Where does the restriction enzymes bind to the DNA? |
-Restriction enzymes travel in pairs and bind to one strand of DNA each -Restriction enzymes can only cut one DNA strand |
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What are the two types of cuts restriction enzymes can do? |
1) Restriction enzymes can do blunt cuts, where both DNA strands are cut evenly 2) Restriction enzymes can also cut to leave sticky ends, where one of the strands extends three or four basepairs further than the other. |
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When a restriction enzyme cuts DNA, what stops the DNA from simply rejoining again? |
-The restriction enzymes take the 5´ phospho group. -DNA Ligase is able to reseal this gap. |
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What is DNA ligase? |
-The molecular glue -DNA ligase can join two pieces of DNA together by forming the missing phosphodiester -This allows the recombination of DNA |
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What are the three features of cloning vectors? |
1) Origin of replication = directs self replication 2) Dominant selectable marker = usually drug resistance of host cell 3) At least one EcoRO (cleavage site) |
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How many plasmid vectors can exist in a bacteria cell? |
Between 1 and hundreds. |
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What are some features of plasmid vectors? |
1) Naturally occuring in many organisms 2) Carries antibiotic resistance genes 3) Can be taken from bacteria, modified and re-introduced into bacteria |
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How many base pairs can a plasmid vector hold? |
Up to 10kbp. |
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What are the features of bacteriophage vectors? |
-A strand of bacteriophage DNA -Central 1/3 is not essential for growth, and can be replaced by foreign DNA |
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How many base pairs can bacteriophage vectors hold? |
Up to 15kbp
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What are the features of cosmid vectors? |
-Cosmids are a hybrid of plasmids and bacteriophages -This combines the best of both -Replicates automatically like plasmids -Infects other bacteria like bacteriophage |
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How many base pairs can a cosmid vector hold? |
Up to 50kbp |
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What are the features of eukaryotic and shuttle vectors? |
-Vectors that are used to introduce DNA to yeast, drosophila, mammals and plants -They can be isolated, mutagenised and re-introduced back into the host |
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What are yeast artificial chromosomes? |
-Yeast artificial chromosomes (YAC) -Genetically engineered yeast mini chromosomes able to accept up to 200-500kbp |
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What are BACs and PACs? |
-Artificial chromosomes similar to BACs but are less complex -They can accept 150-300kbp. |
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What are DNA libraries? |
-A genomic DNA is a set of DNA clones that collectively contain the entire genome of any given organism |
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What is a chromosome-specific library? |
-A chromosome-specific library is the collective set of all DNA in a chromosome |
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What is a complementary DNA library? |
-A complementary DNA library is the collective set of all DNA that is expressed? |
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What are the two kinds of radioactive phosphorus? |
-32P and 33P -They allow us to find the gene of interest |
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What is southern blotting? |
-A technique to find DNA of interest -Radioactive DNA of interest is separated from normal DNA and exposed to X-rays |
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What is northern blotting? |
-A technique to find RNA of interest
-RNA fragments is separated due to size by electrophoresis in an agarose gel -RNA is transferred to membrane to be washed off and the radioactive is separated |
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What is western blotting? |
-Proteins are separated using polyacrylamide gel electrophoresis -Proteins are transferred from the gel to a membrane -Individual proteins can be detected using specific antibodies |
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What is the human genome project? |
-Launched in 1990 -Estimated $3 billion -The goal was to sequence the human genome |
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What were the two methods used in the human genome project, and how did they work? |
-The shotgun: DNA fragments are inserted into plasmids and cloned into bacteria -fragments sequenced using automated systems -Map-based approach: chromosome is cut and ligated into YACs and BACs to create libraries |
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What is Polymerase Chain Reaction (PCR)? |
-Used to amplify large quantities of target DNA -50-10, 000 bo -Used for gene analysis and cloning -Can be used on a suspects DNA at a crime scene -Does not require highly purified DNA |
