TMC12 Flashcards
What is a genome library?
A library of fragments representing the entire genome
What is a cDNA library?
mRNA converted into DNA using reverse transcriptase
Combination of cloned cDNA fragments inserted into a collection of host cells which constitute some portion of the transcriptome of the organism
What is DNA ligase and how does it work?
Joins DNA molecules together by ligating nicks in double stranded DNA
Very specific and can only join 2 DNA molecules when the 3’ OH of one molecule is immediately adjacent to a 5’ phosphate of the other molecule in a double stranded DNA context
What is a restriction enzyme and explain how the restriction enzyme EcoRI cuts DNA?
Cuts DNA, cleaves foreign DNA
EcoRI cuts double stranded DNA at the sequence GGATCC
Cuts at sticky ends - cleaves DNA that leaves 5’ to 3’ overhanging sites
Explain how two DNA fragments can be cut at a specific sequence and joined together.
Use restriction and ligase
What is a plasmid and its essential features?
Small circular DNA molecules in bacteria that are Vehicles used for carrying inserted foreign DNA
Typical features are E. Coli promoter, Origin of replication, antibiotic resistance gene, restriction sites for restriction enzymes
Explain how a piece of human DNA can be inserted into a plasmid.
Restriction enzymes to cut DNA samples and and also cut plasmid DNA
Incubate the cut DNAs together in the presence of DNA ligase.
The ends of the cut DNA anneal and are joined covalently together by the DNA ligase
Explain how restriction enzymes are used in recombinant DNA technology.
Cut DNA samples and plasmid DNA to generate sticky ends
Explain how DNA ligase are used in recombinant DNA technology.
The DNAs are incubated together in the presence of DNA ligase which anneal the cut DNA together at the sticky ends and are joined covalently together by DNA ligase
Explain how reverse transcriptase are used in recombinant DNA technology.
Converts mRNA into cDNA so that introns can be removed
DNA splicing
Explain how plasmids are used in recombinant DNA technology.
Inserted into E. Coli
Plasmid DNA with the insert can be isolated easily from E. Coli cells
Foreign DNA it possesses is cloned
