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1
Q

recombinant DNA

A
  • is regulated
  • NIH Office of Biotechnology activites
  • Institutional Biosafety committees
2
Q

restriction enzymes

A
  • bacteria evolved restriction/modification systems to protect their own DNA from alteration
  • cut foreign DNA, while modifying their own DNA to protect it
  • two components to the restriction system: restriction enzyme & methylase
  • site specific endonucleases
  • blunt ends, sticky ends
3
Q

gel electrophoresis

A
  • separates DNA fragments according to size
  • gels are molecular sieves with different pore sizes
  • different types of gels allow separation of different sizes of DNA fragments
4
Q

molecular cloning of fragments of DNA

A
  • a way to isolate specific DNA fragment
  • the fragment is purified away all of the other fragments
  • the fragment is amplified to create many copies for analysis
5
Q

steps in molecular cloning of DNA

A
  • cut DNA with a restriction enzyme
  • ligate DNA to a cloning vector, using DNA ligase
  • introduce the ligated DNA into a host bacterium
  • detect the desired recombinant clone
6
Q

reasons for DNA cloning

A
  1. isolate an individual gene
    - to characterize it
    - to express the gene product
    - to mutate it in defined ways
  2. to isolate all of the DNA/genes of an organism
    - for DNA sequencing
    - to create a collection of individual genes/DNA fragemnts
7
Q

genomic library

A

a large number of recombinant plasmids that collectively contain the entire genome of an organism

8
Q

how to find a gene of interest in the library

A
  1. pre-genomic era:
    - hybridization and related strategies
    - goal is to find one gene
  2. genomic era
    - DNA sequencing
    - goal is to identify all clones in the library
9
Q

dideoxynucleotides

A
  • key to enzymatic sequencing by synthesis
  • inhibit chain elongation
  • used in ESanger method
  • DNA to be sequenced is used as a template for synthesis in the presence f ddNTPs
  • a nested set of synthesis products is produced in each reaction due to the presence of ddNTPs