Chapter 2: How to Clone a Gene: An Overview

Cloning: asexual reproduction of genetically identical organisms (generation of organisms contains specific DNA)

The Process of Cloning

  • Restriction Enzyme Digest to cut vector (plasmid BAC YAC) and fragment, ligation (T4 DNA ligase) to form recombinant molecule, ligation products introduced into host cells via transformation

Isolation of Genomic Fragments by Library Screening

Gene Libraries give a means to isolate specific cloned fragments, library is created by ligating each of the restriction fragments of a genomic digest into a separate vector and transformed into colony so every fragment in genome is cloned

  • Transformants are screened with modified fragments specific for a target sequence called probes
  • Probed detects target in a hybridization reaction due to complementary base pairing
  • Probes design: complementary, specific, vague for family
  • Probes created invitro DNA synthesis reaction requiring
  • DNA Template (denatured, something for the probe to specify to)
  • 4 deoxynucleotide triphosphate (dNTP); one radioactive (detect probe once synthesized)
  • Buffer containing Mg (allows DNA Pol to work)
  • DNA Polymerase (enzyme to synthesize)
  • DNA Primer (need some info to create, provides start site for DNA POL)

PCR is another way to recover specific genomic fragment, 2 primers so region in between replicates

Genomic Cloning

  • Few weeks maybe longer
  • Need probe
  • Radioactivity

PCR

  • Couple days to receive primers, hours to do PCR if genomic DNA available
  • Need enough info to design primers, no probe

No radioactivity

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