AMB5 Flashcards
(20 cards)
What is a genomic library?
a collection of clones that contain fragments of DNA dervived from nuclei/organelles of a specific organism.
- sum of all inserts carried by vectors
What is a representative genomic library?
all genetic info of organism of interest is contained several times over.
What is coverage?
how many times each gene unit of info is contained in the primary library on average
=av. size of insert X no. of primary clones / size of genome
What is a primary clone?
clones that were produced in the first round of synthesis/prior to amplification
What does the size of the insert depend on?
- DNA quality - large DNA is prone to breaking by shearing forces
- Limits imposed by vector
What is the approx insert size range for a bacteriophage?
10-20kb
what is the approx insert size range for cosmids and fosmids?
30-50kb
what is the approx insert size range for BACs?
100-200KB
What is the approx insert size range for YACs?
1000kb
How is DNA extracted, describe the process?
- cell lysis - sonication/chemicals/physical disruption
- detergents help to separate DNA from other components, in presence of catropic salts like guadnidium chloride.
- phenol denatures proteins. then centrifuge separates into phenol, proteins and supernatand nucleic acids
- to recover DNA add ethanol and centrifuge to get precipitated DNA, spin filaments of DNA that form at the ethanol/water interphase with glass rod.
What does guanidium chloride do?
attacks and denatureds proteins and prevents nucelases acting on the DNA.
After DNA is extracted what must be done to it to prepare it for transition into vector ffor library?
DNA must be sheared and ends prepared compatible to vector. RE used typically
must be split into smaller pieces
Sau3a1 is commonly used (recog 4 bases) end up with v.small DNA pieces. - however in unfavourable conditions can cut randomly at few RE sites. - can choose conditions to get desired size range.
How do you make a phage library?
cohesive ends - cos from phage DNA stick together to create a large concatenamer which is the substrate of the packaging reaction.
lambda dna can be digested to remove internal fragments not essenetial to replication, leaving cos arms.
insert DNA digested with same RE can be inserted between cos arms
package in vitro into bacteriophage lambda particles
= library
Describe cloning in cosmids?
= plasmid that contains cos sequence
similar to phage hoever once DNA injected into host DNA replicates as a plasmid not a phage beause tehre are no phage sequences apart from cos that allow replication.
describe SuperCos?
7.9kb packaging allows 50kb to be pacakgedm limited size of insert.
contains 2 cos sites and other genes for selection purposes like Neo resitance - for mammalian cells and SV40 - ori in mammals
Desribe cloning in fosmids?
why are fosmids better than cosmids for larger DNA
plasmids contain f-factor replicon, instead of ori
f-factor drives replication of small number of copies -ussually one 1.
this is better as mutple copies are likley to be recombined in bacteria. - messing them up
cosmids become unstable due to this
fosmids are therefore better for larger DNA
Describe a BAC vector?
bacterial artificial chromosome.
essentially plasmids that contain an ori, active in e.coli which drive plasmid replication with dynamics similar to bacterial chromosome.- low copy no.
can carry very large inserts without recombination making them more stable.
Describe a YAC vector
yeast artifical chromosome
can mainatain very large inserts.
artifical yeast chromsome with inserted DNA.
How can libraries be screened?
PCR
hybridisation with labelled RNA/DNA
- traddionally screened by idnetifiying clones that contain an insert with specific dna sequence.
solid phase hybridisation - similar to southern blotting
describe solid phase hybridisation?
transfer colonies to nitrocellulose/nylon
lysed using base NaOH and protease
DNA freed is bound to membrane by UV irridation or incubation at high temps
denatured dna covalently bound to membrane by backbone and ss molecules are exposed
labelled complementary dna probes can bind to target dna
