Chapter 17 Flashcards
Recombinant DNA Technology
Recombinant DNA
Recombinant DNA
- Joining of DNA molecules
- Produced by artificially joining DNA from different biological sources not found together in nature
Clones
- recovered copies of recombinant DNA molecule
Recombinant DNA technology
- used to isolate, replicate, and analyze genes
Restriction Enzymes and Cloning Vectors
- Two important tools used to construct and amplify DNA molecules–DNA-cutting enzymes: restriction enzymes–DNA cloning vectors
Restriction Enzymes Cut DNA at Specific Sites
Restriction enzymes
- Produced by bacteria as defense mechanism against bacteriophage
- DNA-cutting enzymes
- Bind to DNA at specific recognition sequence (restriction site) and cleave DNA to produce restriction fragments
- Enzyme cleaves both strands of DNA (digestion)
Recognition Sequences
Palindrome
–Symmetry exhibited by recognition sequences (Nucleotide sequence reads same on both strands).
–Restriction enzymes cut DNA in characteristic cleavage pattern (Figure17-1).
–Sticky ends (cohesive ends): fragments produced with overhangs
–Blunt ends: fragments produced with double- stranded ends
DNA Ligase
DNA ligase
–DNA fragments will seal phosphodiester backbone.
–Joins restriction fragments covalently to produce intact DNA molecules
DNA Vectors Accept and Replicate DNA Molecules to Be Cloned
Vectors: carry DNA molecules
- can replicate cloned DNA fragments in hosts cells
- must be able to replicate independently
- have several restriction enzymes sites to allow insertion of DNA fragment
- carry selectable gene marker to distinguish host cells that have taken them up from those that have not
Plasmids
Plasmids used in DNA cloning
- genetically modified bacterial plasmids - first vectors developed
- genetically engineered to contain: number of convenient restriction sites and a marker gene to select for presence in host cell
Transformation
Plasmids are introduced into bacteria via transformation
- 2 main techniques
1. using ca ions and brief heat shock to pulse DNA into cells
2. electroporation: a brief but high intensity pulse of electricity to move DNA into bacterial cells
DNA cloning
- Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme
- DNA restriction fragments from DNA to be cloned are added to linearized vector in the presence of DNA ligase
- Recombinant DNA is produced and introduced into bacterial host cells by transformation
Selection markers
selectable marker genes
- genes provide resistance to antibiotics
Blue-white selection
- used to identify cells containing recombinant and non-recombinant DNA
- plasmid contains lacZ gene which encodes B-galactosidase
blue-white screening mechanism
Blue - bacterial cells with functional lacZ gene carrying a non recombinant plasmid
White - bacterial cells with recombinant plasmid
Other types of cloning vectors
- phage vector systems
- Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)
phage vector systems
- among earliest vector used in addition to plasmids
- central third of lambda phage vectors can be replaced with foreign DNA without affecting ability to infect cells and replicate
Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)
- Vectors used to clone large fragments of DNA
- BACs are generally very large and circular.
- YACs are linear and have telomeres at each end
DNA libraries
- represent a collection of cloned DNA
- Two main types: genomic and complementary (cDNA)