1-express greater concentration of protein product(compared to eukaryotic cells)
2-require relatively simple media componeents
1-do not perform many important post translational modifications( ex: glycosylation).
2- it is not possible to express large proteins in E.coli.
recognize specific base sequences in dsDNA
break the phosphodiester bonds between 2 nucleotides within the sequence
some enzymes cut the DNA at a different position in the 2 strands,producing a single stranded overhang or a sticky end ex: Eco RI
other enzyme produce blunt-ended fragments with no sticky end. ex: PvuII
sticky end endonucleases
the formation of sticky ends is useful by base pair matching of the sticky ends, any DNA fragment produced by the action of the same restriction enzyme can be joined together in a specific manner.
2 DNA ligases are used in DNA analysis:
DNA ligase from E.coli which requires the nucleotide NAD+ as a cofactor
T4 DNA ligase from T4 phage which requires ATP as a cofactor.
catalalyze the synthesis of a phospho-diester bond between:
1- the 3' -OH group of 1 nucleotide
2- the 5' phoshporyl group of the next nucleotide in a DNA molecule.
ligases are most often used to produce a covalent bond between nucleotides in DNA joined by base pair matching of sticky end
primes are short oligonucleotieds complementary to the ends of the original DNA or RNA strand, are added to the incubatiion mixture.
DNA polymerase I
it attaches to short single standed region in a dsDNA molecule
then it synthesizes a new strand of DNA, degrading the existing strand as it proceeds.
in vitro, it must be incubated at 12-15 Cto prevent more than one round of replication occuringthe DNA polymerase I molecule contains its polymerase and nuclease activity on different parts of the enzyme molecule.
contain the part which retains the polymerase function
it synthesize a new DNA strand complementary to the single strand of DNA( the template) only.
it is used to create blunt ends.
it synthesize a cDNA strand using mRNA as a template
it has its maximum activity at 70-80 C
it remains active up to 90 C
it is used in PCR
DNA modifying enzymes
enzyme that can be used to remove or add groups to the ends of the DNA molecule:
1- alkaline phosphatase which removes a phospate group from the 5'terminus
2- polynucleotide kinase which adds a phosphate group to a free 5' terminus
3- terminal deoxynucleotidyl transferase which add 1 or more deoxynucleotides to the 3' terminus
methods for DNA modification
is a circular DNA duplex
an extrachromosomal segment of DNA found in certain bacteria
is replicated by the cell
contains the requisite gentic machinery, such as a replication origin, to permit their autonomous propagation in a bacterial host or yeast.
why plasmids are good cloning vectors
1- small size (1 to 200 Kb) thus easy to manipulate and isolate
2- circular ( more stable than linear DNA)
3- replication is independent of the host cell
4- several copies may be present ( facilitates replication)
5- frequently have antibody resistance ( detection easy)
steps of rDNA technology or cloning steps
1- identification of the protein to be produced
2- isolation of the gene of interest
3-insertion into a suitable vector (plasmid)
4-insertion of theplasmid into host cell
5-culture ----> clones
6-detection and purification of the desired clone. replicate clone to high numbers
1- are viruses that infect bacteria used as vectors
2- linear dsDNA containg genes are required for viral replication within the host cell.
3- during infection DNA molecules are inserted into the host bacteria
4- 2 modes of replication:
A- with cell lysis
B- without cell lysis
steps of DNA modification using phage:
1-isolation of DNA from phage and digestion with restriction enzymes
2- connection of fragments to foreign DNA using DNA ligase
3- packing of DNA with head and tail proteins
4- infection of bacterial host
5- detection of transferred genes
issues with gene therapy
1-we could genetically engineer any "desirable" trait, but ethically it is un unacceptable
2- we cannot deliver rDNA to human eggs or sperms in order to ensure that all cells have the gene.
3- it can only be done with cells that can be removed form the body, engineered and cultured in vitro and then returend to the body ex: white blood cells. that's why it is called "EX vivo" gene therapy
4- gene therapy of some genetic disease require the delivery of rDNA to different cell types, most of which cannot be easily removed and reintroduced---> will require new technological advances. ex: good cycstic fibrosis gen is introduced via a recombinant adenovirus cia an aerosal spray.
how do you know wheter a disorder is a good candidate for gene therapy?
1- does the condition result form mutation in one or more genes?
2- which genes are involved
3- what do you know about the biology of the disorder
4- will adding a normal copy of the gene fix the problem in the affected tissue
5- can you deliver the gene to cells of the affected tissue.
gene delivery: the key to gene therapy
1- targeting the right cells
2- activating the gene
3- integrating the gene in the cells
4- avoiding harmful side effects
herps simplex virus
when adding a good copy of the gene won't solve the problem.
deal with dominant negative through:
1- a technique for reparing mutations: SMaRT( spliceosome-mediated RNA trans-splicing)
2- techniques to prevent the production of a muatated protein:
A- triplex-helix forming oligonucleotides
RNA ex: HIV
affect only dividing cells.
converted to DNA before activating
S.E: disturb other genes because of intigration randomly
ds DNA: ex common cold
dividing and non-dividing
it will not integrate, gene activation lost after week or 2
S.E: imune response
adino associated viruse
dividing and non dividing
need helper viruse to replicate themselves inside cells
95% integrated at a specific region on chromosome 19 greatly reducing the chance that integration will disrupt the fn of other genes in the cells
NO imune response
herpes simplix virus
ssDNA ex: oral and genetl herps
infect cells of nervous system
don't integrate but remain for long time as a separate circular piece of DNA that replicate when cell divide and it will not disturbe the function of other genes
SE: imune response
circular dsDNA called plasmid
not viral type
no specific target, less effective, no max lenght
will not integrate unless engeneerd
don't induce immune response but some time are toxic