Vector, Clones and Libaries Flashcards
Limitation of plasmid vector
-limited base size <8kb
-transformation with large insertion is inefficient and plasmid with large insert are susceptible to mechanical damage
-hard to maintain and store for long period of time
More suitable vector than plasmid
-Bacteriophage lambda vector 20kb
-high capacity vector 45-2000kb
bacteriophage lambda genome map
-on the left cohesive end: code head and tail
-on the right cohesive end: regulation, immunity, DNA synthesis, lysis
these are needed for the lytic pathway
the middle is the non-essential, coding intergration and exertion for the lysogenic pathway
-total of 49kb, only 40-55 kb can be package into phage head
Modification of lambda into vector
-for cDNA libary: eliminate common restriction sites and remove part of the non-essential lysogenic region (4kb). insert up to 10kb
-for gDNA libary: eliminate common restriction sites and remove entire non-essential region 20kbs. insertion for 10-26 kbs
how to amplify lambda gdna vector
-the removal of common restriction and non-essential make the genome too small to fit into the head
-a stuffer is added , increase the size to 45kb
-the stuffer is removed and purify the left and right arm. add the insert sequence
-only the insert sequence is big enough to fit insdie the head
Why is lambda a good vector
-high efficient at introducing recombinant DNA into cells (induction>transformation)
-larger insert size
-stable and store for a longer period of time
-the higer the capacity, the less clone needed to cover an entire genome
Fosmid vector
-Hybrid of phage and F factor
-Use cos site for phage packaging and ori site for maintenance in bacteria
-Genes associated with the lytic and lysogenic pathway removed
-capabel of 35-45 base pair insert
what does fosmid vector contain
-a selectabe marker
0F-factor and origin of replication
0unique cloning (insertion) site
-cos site (phage genome) phage will consider it as a phage dna
Generating fosmid libary
- the genomic dna is digested and select for 35-45kb fragment, added in with linearized vector
-ligation lead to a concatemer with different dna
-the concatemer is celved at a particular site and is then stuffed into a phage
-fosmid is maintained as a plasmid in a cell
creating a lambda libary
-extration of genomic DNA
-generation of fragements through partial digestion of genomic DNA
-isolate 15kb genomic fragemnt
-isolate the left and right arm of lambda vector
-ligate the dna fragment and vector arms (linear lambda have a cos (cohesive) with 12 bp overhand) which lead to a concatemer
-in vitro packaging into the head
-plate out recombinant phage DNA
the invitro packageing of lambda
-proteins are required for packaging of phage dna into active phage head: protein A,D,E
-endonucleolytic clevage at cos site by protein A generate nicks 12bp apart in opposite strand
-the strain is stuffed into head precursor made up of protein D and E
Amping lambda genomic library
-each phage would contain a different fragment
-mix the phage with host bacteria and plate out
-different colony would contain different fragment
overview of screening using sequence homology
-mixture of nucleic acids are denatured into single strand
-probes are then hybirdise and wash
-However, the nucleic acid are found in the probe head
Step in screening with sequence homology
1/ transfer phage particle into a membrane (the arrangement is preserved) aka plaque lift
2/Release dna from phage and denature it
3/Crosslinking: attach the DNA to the membrane
4/ hybridization- incubate the membrane with probe
5/ wash: removals of unbounded probe
6, identification
gettinf probes
-generate a probe from DNA that has homology to the target sequences
-compare protein sequence and identify region of high conservation
-the probe will be partially complementary
Stringency in hybirdization experiment
-extent of annealing between nucleic acids is determined by stringency: salt concentration and temperature
-in low salt/high temp: high stringency use for detection of identical sequences
-in high salt/low temp: low stringency use for detection of identical and related sequences
Charatecerisation of a libary clone
-locate target sequences by restriction mapping- souther blotting
-analyze the expression of target sequence
-determine the nucleotide sequences
-require mapping and subcloning
southern blotting
1/cleave recombinant lambda clone DNA with a restriction enzyme
2/separate cleaved DNA by gel electrophoresis
3/denature DNA
4/transfer the single strand DNA to a membrane by capillary blotting
restriction mapping
-the digest recombinant clone with different restriction enzymes and separated by gel electrophoresis
-transfer for southern blotting and hybridization with probe
-identify the hybridizing fragments of recombinant clone
-double/triple digest is needed to make a restriction map
-subclone to investigate interested sequences
directional and non-directional subclone
-directional subclone gave different stiky end so they can only be insert in one orientation
-non directional have either the same sticky of blunt end, can be inserted by either oritentation
