Lecture 13 - Recombinant DNA/Biotechnology Flashcards
What is the core genome?
conserved genes - ancestral genes can help identify pathogens
what are mobilomes? list them.
mobile genetic elements
- plasmids
- transposons
- phages
- genomic islands: large genomic segments in a cell’s genome that originated in another species
What the bacterial defenses against foreign DNA?
Restriction-modification systems (cuts DNA up)
CRISPR (think phage DNA “memory bank” almost like immune system antibodies kind of)
What are the restriction-modification systems?
(cuts up DNA)
- these are restrictive enzymes which degrade unmethylated DNA
- they CAN NOT degrade methylated DNA
- their reaction does not require any energy it is energetically favorable
- phage DNA that is unmethylated will be degraded and thus not replicated and passed on
- methylated phage DNA on the other hand can be replicated per usual
- many different enzymes with many different sequences
What is CRISPR?
Clusters of Regularly Interspersed Short Palindromic Repeats
CRISPR array is a section of the genome that holds fragments of broken phage DNA that once invaded the cell
This CRISPR array serves as a “memory bank”
After the CRISPR array/”memory bank” is transcribed it is stored in this machine
the transcribed cRNAs are held in the cas nuclease- crRNA complex
This machine will then destroy incoming DNA - will kill some phage which comes in
- they can also modify the target DNA which is why CRISPR is used as gene therapy
Restriction enzymes
common examples: Alul, Bamhl, EcoRl
- each have recognition sequence which is where the cleavage site is
Recombinant DNA
molecules made by joining DNA from 2 different sources
What happens when you use a vector in genetic engineering?
if DNA is inserted in a vector it is continued through all the future cells and is almost impossible to get rid of
how do you clone DNA in bacteria?
Isolate DNA
use restriction enzymes to generate DNA fragments
cut vector with same enzyme
Join vector with fragments (DNA ligase is needed now)
Introduce recombinant molecule into new host
How would you get multiple copies of genes produced?
High copy number vector + inserted gene = lots of copies of gene produced
What are the properties of an ideal vector?
- Origin of replication
- Selectable marker: a gene encoding resistance to an antibiotic such as ampicillin
- Second Genetic Marker: a gene such as lacZ that encodes an observable phenotype
- Multiple Cloning sites: containing the recognition sequence of several different restriction enzymes
NOT plated on mckary agar
what is X-Gal?
simply a dye indicator
What is a selectable marker?
a gene encoding resistance to an antibiotic such as ampicillin
what is a second genetic marker?
a gene such as lacZ that encodes an observable phenotype
DNA sequencing (in vitro) can be done using what method?
Dideoxy Chain Termination Method
