Cloning & genetic libraries Flashcards
What is cloning?
Copying ____ to produce multiple identical copies:
- organism
- plant
- cell culture
- piece of DNA
Which enzymes are used during cloning?
Restriction endonucleases, DNA pol., DNA ligase, reverse transcriptase, phosphatase
How is an insert prepared for cloning?
a) restriction endonuclease digestion: cut @ specific sites
b) physical fragmentation: then use DNA pol. to fill sticky ends => blunt ends
c) making complimentary copy (ds-cDNA) of RNA using reverse transcriptase: mRNA -> ss-cDNA -> ds-cDNA
d) amplify specific regions w/ PCR
How is a vector prepared for cloning?
- digested w/ restriction enzyme => sticky ends that complements DNA to be cloned
- (OPTIONAL) dephosphorylate termini of vector using phosphate enzyme = prevent recircular w/out an insert
- vector is purified = avoid reagents interering w/ ligase react.
List essential elements of a cloning vector
- Multiple cloning site (MCS): insertion area = allow DNA to be inserted in vector
- Origin of replication: make multiple copies of self (including the inserted DNA)
- Selectable marker gene (antibiotic resistance): make more copies & outcompete others
What to consider when choosing a suitable vector?
properties that will depend on the purpose of cloning and the size of the insert
Why are transformed cells allowed to recover in a rich (selective) media?
cells containing the vector will be able to grow (w/ or w/out inserted DNA) bc vectors have selectable marker gene which resists antibiotics
How can we use blue-white screening to select our desired fragment?
- MCS is in lacZ gene fragment => metabolise X-gal => produce B-galactosidase=> blue colonies
- but inserted DNA in MCS disrupt gene frag. ≠ metabolise ≠ produce => white colonies
What is a DNA library? & name 2 types of libraries
collection of clones that represent a (partial / whole) genome of an organism
- Genomic library
- cDNA library: genes expressed (RNA -> cDNA)
Explain how a genomic DNA library prepared?
- genome cuts into smaller pieces
- Clone pieces into appropriate vector
- Insert recombinant vector in bacterial cells
- Separate colonies w/ diff. genomes
How do we generate complete coverage of a whole genome?
- Partially digest/cut pieces (my limiting no. enzymes or time course)
- overlap pieces of strands to help sequence pieces of DNA in right order
Explain how a cDNA library prepared?
- Reverse transcriptase mRNA -> cDNA
- DNA cloning
* note: highly expressed / active genes will produce more copies
What is the purpose of a probe?
Identify a gene during cloning
What can be used for a probe?
short ssDNA
What’s the difference b/w genetic engineering & DNA cloning?
Engineer: change DNA
Cloning: make multiple identical copies