Hunting the Gene Flashcards
What are the three methods of horizontal gene transfer?
Transformation
Conjugation
Transduction
Phage mediated
What is transduction
Phage-mediated DNA transfer
- bacterial chromosones may be incorperated into phade particles
- when the phage attacks its next bacteria, it can insert this bacterial DNA into the new one
What is conjugation?
Plasma mediated transfer (ex: plasmid F)
- F-pilli form between two cells
- allows a large, low copy plasmid to travel between cells
- f-plasmids can also insert themselves into the chromosone of other cells (HFr) or just a few genes (F’)
What is transformation?
Uptake of naked DNA from the envrironment
- many bacteria can take up DNA from teh envuronment and incorperate it into their genome by recombination
How does gene cloning work?
Uses smaller, large copy plasmids
- Cut plasmids and DNA with same restriction endonucleases (EcoRI)
- Hybridisation by DNA ligase
What are some features of a plasmid that faciliatate gene cloning?
- Replication origin that allows it to be replicated in bacterial host
- Selectable marker (eg. antibiotic resistance)
- helpful if its insertion disrupts a gene to produce a detectable phenotype
How may we overcome the problem that DNA sequences are hard to incorperate by plasmids?
Cloning vectors based on phage
- phage gamma is very popular as its central genome is not required for replication
- therefore can incorperated into bacteria by phage infectoion
- allows cloning of DNA fragements 10-15kB (plasmids only allow 10ish)
What is a genomic library?
A collection of cloned DNA fragments that represent the whole genome of an organsism
- usful for popular cloning vectors such as phage lamda
How might a genetic library be created
…
How might we clone even larger fragements than with phages?
Cosmids - hybrid of phage and plasmid
allows cloning of up to 40kb
Artifical chromosones - up to 1000kb
