Recombinant DNA Technology-cloning- Screening Abd Genomic Flashcards
What are the steps of molecular cloning?
- DNA fragments to be cloned are created by digesting DNA with restriction endonucleases
- A vector, which replicates autonomously, is selected to act as a vehicle to transfer the fragment into a host cell *digest it with the same restriction endonucleases
- The fragments are then lighted into the vector
- The vector/fragment hybrid, called a recombinant DNA molecule, is introduced into a host cell
- Recombinant DNA will replicate making many copies of itself AKA —> clones
- When host cell replicates, the daughter cells also contain the recombinant DNA, forming a colony of identical cells
- Recombinant DNA is recovered from the colony (the larger the colony—> the higher the yield of cells) and can be purified and analyzed
What are four important factors in choosing a vector?
- They must be able to self-replicate both before and after insertion of foreign DNA
- Must have a region called a Multiple Cloning Site (MCS)
- A number of unique restriction sites close all in the same region which are unique and not present anywhere else in the vector
- Must contain a selectable marker
- Typically a gene which confers antibiotic resistance and is not found in the host cell
- Sometimes it is a gene for an enzyme that is not found in the host cell
- Need to recover the recombination DNA from the host cell easily
What is plasmid ?
Naturally occurring, circular, double stranded DNA that is extra-chromosomal (not part of the genomic DNA)
- Must contain origin of replication (ORI) and replicate autonomously when it is contained inside a bacterial cell
- High copy number(> 500 copies made per cell)
- Antibiotic resistance gene to select bacteria which have taken up the plasmid
What is a multiple cloning site?
Multiple cloning site (MCS or polylinker region) containing numerous UNIQUE restriction sites for inserting similarly cut fragments of DNA
What is the reporter gene?
Reporter gene or a-complementation to select bacteria which have taken up plasmid that has a foreign DNA insert
What has plasmid been genetically engineered to do?
We have genetically engineered plasmid and we put it in a genetically engineered bacteria
-This plasmid with LacZ gene is put in a bacteria with LacZ deleted
Explain the Blue/white reporter gene
- Plasmid contains complete LacZ (host cell has LacZ deleted from genome)
- Functional enzyme made if empty vector is taken up by engineered cloning cell
- Bacteria are grown derivative of GALACTOSE, called X-GAL, that turns blue when cleaved by a functional B-galactosidase protein
If the bacteria turn blue, they have empty vector… Don’t bother amplifying and purifying this vector
Describe the blue/white reporter gene
- Plasmid with intact LacZ gene allows cell to metabolize X-gal and form blue colony
- Multiple cloning site of plasmid cut with restriction enzyme
- DNA to be cloned cut with same restriction enzyme
- Recombination plasmid cannot metabolize X-gal and will form white colony
- DNA insertion disrupts LacZ gene, the transform bacteria with plasmid
What are competent cells? How are they formed?
- competent bacterial cells are altered cells so that DNA passes through cell wall
- E. Coli cell walls are altered with treatment of calcium chloride and/or rubidium chloride
- Foreign plasmid DNA associated with the cell exterior
- Foreign DNA is taken up by competent bacterial cells by brief heat shock treatment by being placed in 42 degrees Celsius for 30-60 seconds
- Cells are gently spread on nutrient agar plates, each cell will form a colony in a plate
How to amplify and purify plasmid over days?
- Lyse cells, extract DNA
- Treat with ethidium bromide
- Add to solution of CsCl and centrifuge
- CsCl forms density gradient. DNA settles according to its density
How to amplify and purify plasmid over minutes?
Isolate bacterial cells and spin, add RNAse A to pellet cell. Add detergent and NaOH then add acetic acid
Discard pellet and transfer supernatant—> binding principle of QIAGEN Resin (DEAE-diethylaminiethanol)
After binding, spin, wash, spin then elute and spin to receive purified DNA
Summarize amplification and purification of plasmid
A positive colony is selected to inoculate liquid growth media (all work done aseptically as not to start growing stray bacteria)
- 17-24 hours later culture will contain trillions of identical cells with your recombinant DNA ready to harvest
- Easy to purify plasmid DNA these days, with resin that binds
Describe bacteriophage(gamma) as a vector
- Can hold larger pieces of DNA than plasmid vectors
- Often used for preparing genomic or cDNA libraries
- Up to 20kb of the dispensable region can be cut out and replaced with foreign DNA (not needed for lytic cycle)
- Vectors have been “disarmed” so they only grow under lab conditions
Explain how bacteriophage gamma acts as a vector
- Central gene cluster is removed by restriction digest
- Foreign DNA is cleaved by same restriction digest
- Ligation of foreign DNA to the left and right arms of phage DNA
- Recombinant DNA is mixed with phage proteins and is packaged
- Recombinant particle infects bacteria growing on an agar plate “transduction”. Recombinant gamma particle infects bacteria growing on an agar plate “transduction”.
- Cells that take up recombinant particles lyse and look like a clear spot on the agar plate containing a lawn of bacteria, also called a “plaque”
- Each plaque contains millions of recombinant phage particles
- Newer phage vectors also have blue/white selection
What are artificial chromosomes?
They can be used as vectors
- used for mapping and analyzing eukaryotic genomes
- Human Genome Project(sequencing the entire genome) used both Bacterial (BAC) and Yeast (YAC) artificial chromosomes
- Used because they can hold >300 kB inserted DNA
- Also to investigate sets of genes that are adjacent to one another
Describe BACs as vectors
BACs are made using a bacterial plasmid called “fertility factor” or F factor
- This factor promotes the even distribution of plasmids after bacterial cell division
- Can hold up to 350 kB of foreign DNA
Bacterial F’ plasmid is cut within LacZ gene with restriction enzymes + gene of interest is cut out using restriction enzyme
F’ plasmid and gene of interest ligase
BAC is electroporated into E. Coli cells
Cells are plated on X-gal/IPTG medium
White colonies are composed of transformed LacZ- cells
