Recombinant DNA Technology-cloning- Screening Abd Genomic Flashcards
(40 cards)
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
Describe YACs as vectors
YACs are kept circular till digested by restriction enzymes to insert foreign
-Can hold up to 1000kb
They contain:
-Origin of replication called ARS for autonomously replicating sequence
-A centromere to ensure that there is segregation into daughter cells
-Telomeres for stability of the chromosome ends
-Selectable markers on each arm
Typically URA3 which encodes enzyme for biosynthesis of uracil and TRP1 which encodes enzyme for biosynthesis of tryptophan
One marker on each side of insert so we know when we make a complete YAC
Describe DNA Libraries
Vectors used to assemble a library of DNA fragments
To study the entire genome of an organism
DNA fragments are generated by restriction digest
Fragment then ligated into vectors
Recombinants are transferred into host cells (one recombinant per cell)
Each recombinant can be grown (amplified) to yield an abundant and pure segment of genomic DNA(easier to study, sequence etc.)
Collection of recombinants are probed and characterized
Fully characterized library can be readily retrieved whenever a new question is asked
Two main types: genomic and cDNA
Describe partial digestion of genomic DNA
Partial digestion of identical pieces of DNA can result in a series of overlapping DNA fragments or contigs
This strategy is used in sequencing and reconstructing large piece of DNA or whole genomes
How to purify DNA by size -gel electrophoresis ?
Principle: DNA is a negatively charged molecule, when placed in an electric field, will migrate toward the positive pole
- DNA is separated exclusively by size within a matrix (like a gel) using electrophoresis
- Smaller pieces will migrate faster though the matrix than larger pieces
- Gel is a thin slab with wells to load the sample
- Gel is immersed in a buffer with ions and buffer to maintain the pH
- Agarose separate DNA fragments from 100 bp- 50,000 bp(or 50kb)
Describe agarose electrophoresis
Agarose is a polysaccharide from seaweed
-DNA sample is mixed buffer containing high density liquid, dyes to follow the migration of your sample and ethidium bromide to visualize your DNA fragments under UV light
What is the genomic library?
-DNA fragments representing the whole genome of an organism
-First digest the DNA into convenient sizes(15-20 kB for phage gamma)
Partial restriction digest is typical
-Use limiting conditions so that a recognition site is cleaved just occasionally
- Generated continuum of overlapping DNA fragments
-Helps ensure full coverage of genome
-Purify fragments by size (selecting those which are appropriate for your vector)
Can calculate how many fragments (clones) you need for full genomic civerage
Describe the size of the genomic library
Human genome is. 3x10^9 bp( 3 billion)
- divide by 20,000 bp fragments
- need atleast 10^6 (million) clones for humans
- Always add a few more to be sure
What is the principle of making a library of expressed genes?
Principle: mRNA is isolated from particular tissue, cell type or even developmental stage
mRNA not cloned directly
cDNA must be generated from mRNA
