LECTURE 5 ( Genomic Libraries) Flashcards
What is a genomic library?
A simple collection of clones that contain fragments from the entire genome of an organism.
What is the coverage?
how many times each gene is contained in the library. calculated multiplying the average of insert size x number of clones.
What is a primary library?
A library formed before amplification
What are the two quality parameters of a library?
insert size ( that depends in many factors; quality f DNA material, the larger the DNA the easier to break by normal forces e.g pipetting) number of inserts
How can we extract DNA from cells?
we need to centrifugate to separate solubles, the we add phenol and centrifugate again. we will get the NA on top, we get it with a pipette and add it to a tube that has ethanol and a glass bar, the DNA is precipitated around it and the rest of the stuff come to the bottom.
What are the main 5 types of libraries?
- bacteriophage
- cosmids
- fosmids
- Bac
- Yac
What are bacteriophage libraries?
you get your inserts from a eukaryotic organism, then you get the vector from a bacteriophage , they’re ligated and from a very large molecule arms + inserts.
That plasmid is inserted into a bacteriophage that will infect ecoli
What are cosmid libraries?
the same procedure, cut a piece from a eukaryotic DNA (insert) and insert the insert into a vector that will be inserted into the bacteriophage as a plasmid however this time when the phage infects the bacterium the clone will be inserted as a plasmid bc comes from a cosmid
Why are the bact not infected when bacteriophages are used for cloning?
because apart from the cos seq nothing can allow th e replication of the vector because it has been extracted.
What is a common cosmid vector for cosmid libraries? how big is it and how big inserts does it allow?
Supercos it’s 7.9kb big
it allows up to 40kb inserts
What’s the MCS of Supercos 1? what is NotI for?
EcoRI NotI T3 BamI T7 NotI EcoRI
NotI allows the insert to be excised later.
How many oris does Supercos1 have?
It has 2 oris one for mammals (SV40) one for E.coli (bt the two antibiotic resistance)
Does Supercos 1 have antibiotic resistance genes?
yes it has one for ampicilin and one for neomycin.
What is a fosmid library?
extract euk DNA use fosmid as a vector insert it into phage infect bacteria
What is a common BAC vector? Why can it be used to reduce the number of empty vectors?
pBACe3.6: It has the SacBII gene that encodes saccharine that it’s expressed when there’s no insert therefore lethal for E.coli when there’s sucrose so sucrose can be used as a negative marker to reduce the number of empty vectors.
Which one is the hardest vector to make libraries with?
YAC
What is the ‘Chromosome walking’ method for?
It is used to know which clone we should chose, its an overlapping library.
Out of all the vectors? which one can we chose?
depends on many facts; size of the insert
At what conditions are libraries kept?
normally -80ºC in glycerol for plasmids and cryoprotected for bacteriophages
How can we know how good a library is?
you select a number of clones and grow them individually. extract the plasmid and run a gel and the bands of the gell will tell you whether the plasmid has the vector or not
What are M13?
they’re a type of bacteriophage that infects e.coli and its sex specific (it attaches to the pili and only pili in bact that have the f plasmid.
What does it mean that a library contains the whole genome of an organism? How is a genomic library forme?
It means that you get the genome and cut it into different pieces of different sizes (this is random). then we insert them into a plasmid or a phage using cohesive ends and the vector will insert into a bact and the bact makes several copies we lyse the bact cells and collect the copies to create a library then we can screen the library
What if we want a specific gene of an organism? why is a library useful here?
Because we make a library of the whole genome and then we screen to find our gene of interest.
How can we find a gene of interest in a genomic library?
by screening the library.
How is a library screened?
You have a petri dish with non-infected bact, you infect them with phages that contain different inserts of the genome. you take nitrocellulose stick to the dish and get the bact there. then you lyse the cells and the DNA will remain bound to the nitrocellulose, then you use labelled probes that are complementary to the seq of interest. give a wash and many non-hybridized DNA will come off, then you plate it and the fluorescent ones are the pieces of interest that you can then collect.
What is a “representative” genomic library?
Library that contains all the DNA of the organism
by which 4 methods can genomic libraries be screened?
- PCR
- radiolabelled DNA probes
- radiolabelled RNA probes
- fluorescently labelled oligos
