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
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
cDNA library starts with isolated mRNA…
cDNA contains only coding region of the expressed gene(no introns or regulatory regions)
- mRNA contains a polyadenylated 3’ tail which lets us isolate it from other RNA species
- First strand synthesis of cDNA using reverse transcriptase and DNA polymerase and a primer (poly-T)
Reverse transcriptase tends to favor creating a hairpin loop and the end of the mRNA making a natural primer
- Second strand synthesis of cDNA takes advantage of this loop
- Add Klenow fragment of DNA polymerase (5’ to 3’ polymerase with 3’ to 5’ proofreading)
- Add S1 nuclease which cleaves ssDNA hairpin structure
- Now have double stranded DNA molecule to clone into vector
- cDNA library of expressed
Summarize cDNA vs genomic library
The cloning of a population of genomic DNA fragments results in genomic DNA library
-The cloning of a population of cDNAs results in a cDNA library
What does screening/screening a library mean?
To search DNA sequence in a library
You need an appropriate probe
What is a probe?
Probe is usually a small piece of DNA that is same or similar to the sequence you are looking for
Probe is altered so you can detect it:
-Usually by radioactive label at the end of the sequence
-Moving toward fluorescent markers now
What are the types of probes?
Heterologous probes
Homologous probes
Degenerate probes
EST probes
Degenerate probes
What are heterologous probes?
A known gene used to find unknown gene like using mouse sequence to pull out human sequence of a particular gene
What are homologous probes ?
Exactly complementary to sequence of interest
-May organisms have completely sequenced genomes and oligonucleotides are made directly
What are degenerate probes?
Using the protein sequence, all possible oligonucleotide combinations are used
What are EST probes?
Expressed sequence tags: partial cDNA sequences from data banks created from quick sequencing of a cDNA libraries, only 5’ or 3’ ends and the protein product is inferred from sequence
Using the protein sequence, all possible oligonucleotide combinations are used
What is the function of labeling probes?
To make macromolecules visible to the unaided eye
Explain what is random priming
Incorporating radioactive nucleotides along the length of a fragment of DNA
-DNA denatured and random hexamers are annealed
-Add NTPs and Klenow fragment of E. Coli DNA polymerase 1
This technique gives the most radioactive prove as many labelled dNTPs are incorporated
What is in vitro transcription?
RNA is labelled while being transcribes from a cDNA template
-Transcription from promoter region using appropriate polymerase (depends on vector)
-Add labeled and unlabeled NTPs
What is klenow fill-in?
DNA fragments are generated by restriction digest which creates SS 5’ overhangs
-Klenow fragment of E. Coli DNA polymerase 1 will fill in gaps with labeled & unlabeled dNTPs
What is the purpose of labeling probes? What is one type of labeling?
- To make macromolecules visible to the unaided eye
- Oligonucleotide 5’ end labeling: where terminal (32-gamma phosphate of ATP) is added to the 5’ end of probeEnzyme is T4 polynucleotide kinase
What is colony hybridization?
Involves replicating colonies on to a durable membrane prior to hybridization with a labelled nucleic acid probe
What are the steps of colony hybridization?
- Plating bacteria containing library-transferring bacteria colonies onto membrane
- “Cracking” bacteria to releases denatured DNA
- Hybridization of membrane with radiolabelled probe
- Exposure of membrane to X-ray film
- Inpdentification of specific clone containing DNA of interest
