chapter 6 Flashcards
recombination
joining DNA from one DNA molecule (donor) to another (recipient)
also called horizontal transfer when the donor DNA comes from the same/different species
homologus recombination
-most common
-similar to crossing over
long regions are broken off and recombined leadin to crossing over
it is carried out by enzymes like RecA
site-specific recombination
differs from homologus recombination by
-occuring on smaller and more specific sites.
-catalyzed by enzymes called Recombinases
this is how donor DNA is incorporated into a hosts DNA molecules
3 ways of horizontal gene transfer
- Trasformation (directly from the environment) the cell must be competent
- Conjugation (transfered from a donor cell)
- Transduction (transported by a bacteraphage virus that has another cells DNA by accident)
-generalized Transduction - only host DNA transfered by lytic phages
-specialized Transduction - still has some phage gene but some host DNA transfered by lysogenic phages
conjugation (F+, F-, Hfr, F’, lac operon)
the lac operon is the gain of another hosts genes. This is accomplished by transfer of F’ or Hfr transfer. F’ is when the F+ has some of the hosts DNA attached to it and goes into another cell. Hfr transfer is when the F+ is fully integrated into the host genome and goes across to the other cell (takes longer and somtimes doesnt fully complete)
F- just means u dont have the gene to create pilus for conjugation
vesiduction
transportation of DNA through vessicles
simple tranposition
cut-paste transposition
transposase catalysis excision of the MGE, followed by cleavage of the new spot and insertion from ligase
replicative transposition
copy-paste
MGE remains at its spot, and a copy is made and pasted somewhere else
MGE’s
mobile genetic elements can inflence gene function by causing mutations or by providing regulatory sites that alter expression patterns of the adjacent genes. example: antibiotic resistance
transposition
shifting segments of the genome
restriction enzymes/endonucleases
they cut DNA at specific sequences
they recognize palindromic sequences
they can cut blunt or stagered
5’ GAATTC 3’ = 5’ G AATTC 3’
5’ CTTAAG 3’ 5’ CTTAA G 3’
gel electrophoresis
used to separate DNA fragments by size and molecular weight. Smaller ones move through the gel faster
cDNA cloning/synthesis
Complimentary DNA is used to make DNA from mRNA. Usually is goes DNA -> mRNA
However, cloning eukaryotic DNA into bacteria can’t work because bacteria cant splice out introns.
So mRNA is reversed transcribed into usable cDNA without introns for bacteria. The resulting cDNA can be cloned without need for RNA processing.
copies can also be used to store as permanent collections in cDNA libraries.
cloning vectors (4 types)
a cloning vector is a small peice of DNA taken that can be stably maintaine in an organism and into which foreign DNA can tag along to be cloned
(ELECTROPORATION and TRASFORMATION allow these things to get through the membrane)
Plasmids –>
advan: self replicates, easy to handle
limitation: small DNA insert size
λ Phages/Viruses –>
advan: easy to use, high transformation effiecincy
limitation: small DNA instertion size
(hybrid) Cosmids = plasmid + λ phage –>
advan: larger fragments
limitations: phage packaging limitations, unstable inside E.coli
Artificial Chromosomes –> BACs and YACs
advan: MEGA fragments are large as fugg
limitations: YACs (yeast) are bigger but less stable than BACs (bacterial)
what are the minimum requirements to be a cloning vector?
- origin of relpication
- restriction sites called multicloning site
- selectable marker
