Cloning & genetic libraries Flashcards

1
Q

What is cloning?

A

Copying ____ to produce multiple identical copies:

  • organism
  • plant
  • cell culture
  • piece of DNA
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2
Q

Which enzymes are used during cloning?

A

Restriction endonucleases, DNA pol., DNA ligase, reverse transcriptase, phosphatase

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3
Q

How is an insert prepared for cloning?

A

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

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4
Q

How is a vector prepared for cloning?

A
  1. digested w/ restriction enzyme => sticky ends that complements DNA to be cloned
  2. (OPTIONAL) dephosphorylate termini of vector using phosphate enzyme = prevent recircular w/out an insert
  3. vector is purified = avoid reagents interering w/ ligase react.
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5
Q

List essential elements of a cloning vector

A
  • 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
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6
Q

What to consider when choosing a suitable vector?

A

properties that will depend on the purpose of cloning and the size of the insert

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7
Q

Why are transformed cells allowed to recover in a rich (selective) media?

A

cells containing the vector will be able to grow (w/ or w/out inserted DNA) bc vectors have selectable marker gene which resists antibiotics

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8
Q

How can we use blue-white screening to select our desired fragment?

A
  • 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
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9
Q

What is a DNA library? & name 2 types of libraries

A

collection of clones that represent a (partial / whole) genome of an organism

  • Genomic library
  • cDNA library: genes expressed (RNA -> cDNA)
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10
Q

Explain how a genomic DNA library prepared?

A
  1. genome cuts into smaller pieces
  2. Clone pieces into appropriate vector
  3. Insert recombinant vector in bacterial cells
  4. Separate colonies w/ diff. genomes
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11
Q

How do we generate complete coverage of a whole genome?

A
  1. Partially digest/cut pieces (my limiting no. enzymes or time course)
  2. overlap pieces of strands to help sequence pieces of DNA in right order
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12
Q

Explain how a cDNA library prepared?

A
  1. Reverse transcriptase mRNA -> cDNA
  2. DNA cloning
    * note: highly expressed / active genes will produce more copies
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13
Q

What is the purpose of a probe?

A

Identify a gene during cloning

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14
Q

What can be used for a probe?

A

short ssDNA

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15
Q

What’s the difference b/w genetic engineering & DNA cloning?

A

Engineer: change DNA
Cloning: make multiple identical copies

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16
Q

5 steps for cloning

A
  1. obtain desired DNA fragment
  2. Prepare suitable vector
  3. ligation of fragment (insert) to vector
  4. transformation of cells w/ recombinant vector (vector put in cell)
  5. Screen the transformed colonies w/ recombinant vector