Biotechnology and Recombinant DNA Flashcards
What is Biotechnology? What are examples of products formed?
Biotechnology: The manipulation of living organisms, or cell components to produce useful products
-Foods, antibiotic, vitamins, enzymes
-Pest resistant crops
-Bacterial stains for waste treatment, environmental oil clean-up
-Limited to a cell’s own products until the 1980s.
What is Recombinant DNA (rDNA) technology/genetic engineering?
Recombinant DNA (rDNA) technology/genetic engineering: procedures used to join DNA segments together in vitro.
What is a clone? Describe the two definitions
What is a clone?
1) Population of cells arising from a single parent cell
2) Processes used to create copies of DNA fragments
Describe what gene cloning is and how the process works. What is another way to obtain gene of interest?
Gene cloning: the production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques
**All steps in process to ligate and recombine DNA all occur from E.coli
Process:
1. Vectors, such as a plasmid is isolated (from Bacterium; E.coli)
2. DNA (containing gene of interest) is cleaved by an enzyme into fragments
3. Gene is inserted into plasmid
4. Plasmid is taken up by a cell such as a bacterium (almost always E.coli)
5. Cells with gene of interest are cloned
-The goal may be to make copies of gene OR make protein product of gene
-Another way of obtaining gene of interest:
PCR (polymerase chain reaction)
most bacterial cells have many copies of plasmids (not just one chromosome and one plasmid)
Describe the many applications of gene cloning that occur. Discuss one where gene is of interest vs product of gene is of interest. What are examples?
Applications of gene cloning
-it may be necessary to isolate and transform plasmid with a cloned gene into another cell type
-If the gene itself is of interest.
-Plasmid broke genes are Easily manipulated (change nucleotides or fuse two genes together)
process: copies of gene can be harvested Ex: gene for pest resistance will be inserted into plants. Also, the gene alters bacteria for cleaning up toxic waste.
-The product of the gene is of interest
-Cloning human growth hormone was an early success.
process: Cells make a protein product. The desired proteins are harvested
Ex: Amylase, cellulase and the enzymes prepare fabrics for clothing manufacture
-Human growth hormone treats stunted growth
Differentiate between Natural vs Artificial selection. Provide examples
Microbes can be isolated from natural environments that produce a desired product
-Natural selection: Organisms in nature with characteristics that enhance survival are more likely to survive
ex: plumage of peacock, mouse blends in with environment, lizard’s long legs (run faster), Arctic fox has white fur (blend with habitat)
-Artificial selection: humans select desirable breeds of animals or strains of plants
- A farmer chooses high milk producing cows for breeding
-Pure bacterial cultures with favorable characteristics can be selected
-Beer brewing (efficiency, taste, alcohol (content)
-antibiotic producing bacterial strains (also elevated expression)
how can new strains be created?
New strains can be created by exposing microbes to mutagens or directly manipulating their DNA
(mutagens like base analogs)
Differentiate between Random mutagenesis and site-directed mutagenesis
Random mutagenesis (mutagen exposure) can be used to increase the chances of obtaining a desired strain and mutants identified by selecting or screening
(increase rate of mutation in cell and randoms genes mutagenized)
-irradiating fungus generated a strain that produced 1000x penicillin
Site-directed mutagenesis; is a method to create specific, targeted change, in double stranded DNA
(ex: change one nucleotide to another, or delete 3 genes, insertions)
What are Restriction enzymes? What is their function? What kind of sequences do they deal with?
Restriction Enzymes (type II/useful for rDNA technology)
-Restriction enzymes are Endonucleases that cut DNA at defined positions, close to or within their recognition sequences
-typically recognize 4-, 6-, 8- base sequences (**cutting frequency*)
(6, 8 base sequences are more useful for cloning)
-Cut sequences the same way each time
-some produce Blunt ends (cut on both positions on same strand), others produce staggered (Sticky) ends
-Staggered ends can be used to join two pieces of DNA with complementary ends
-Most restriction enzymes recognize palindromic sequences
(EcoRI: has 4 base overhang)
explain the derivation of the EcoRI name and what each letter means.
Derivation of EcoRI
E: means Escherichia ; describes genus name
co: means coli; describes species name
R: means RY13 ; describes strain (if needed)
R is first letter of strain
I: means first identified; describes order of identification in bacterium
Explain how restriction enzymes are used in recombination DNA methodology
Restriction enzymes are used in recombinant DNA methodology
1) Restriction enzyme cuts double-stranded DNA at its particular recognition sites
2) These cuts produce a DNA fragment with two sticky ends
DNA from another source, perhaps a plasmid, cut with the same enzyme
3) when two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing
4. The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur.
5. The enzyme DNA ligase is used to unite the backbone of the two DNA fragments, producing a molecule of recombinant DNA
(DNA fragments with blnt ends can ligate to one another)
How are sticky ends of DNA fragments ligated? What about DNA fragments with blunt ends?
When you have sticky ends, those overhangs have to be complementary to another overhang of the same length
- Blunt end fragments can ligate to any other blunt end
Blunt fragments cannot ligate to sticky end.
(ligase can ligate two blunt ends together)
What are the recognition sequences for enzymes Sal/I and Xhol? Can either starting enzyme recut the ligated the DNA ?
Compatible cohesive ends
-Sa/I and Xhol are enzymes that can cut DNA sequences and then they can be ligated back together
Sal/I Xhol
–GTCGAC– –CTCGAG–
–CAGCTG– –GAGCTC
-these sequences have complementary overhangs
Cutting using both enzymes produces
–G TCGAG–
–CAGCT C–
Which can then be ligated back together:
–GTCGAG–
–CAGCTC–
Notice hybrid sequence for Sal/I starts with G and ends with G meanwhile original sequence started with G and end with C (hence they are different)
Can Either stating enzyme recut the ligated the DNA
NO, it depends on the two enzymes that you have and whether new hybrid sequence will be cut.
REVIEW THISS
Why do bacteria produce restriction enzymes? How is Host DNA modified and explain why?
** They RESTRICT the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host bacterial cell by cleaving it.
(Bacteria produce restriction enzymes as a Defense mechanism against incoming viruses)
The host DNA is MODIFIED by methylation of their sequences at C or A nucleotides
A is more prevalent
-This modification protects the bacterial host DNA from degradation by its own restriction enzyme
-called Restriction modification system (Methylase + endonuclease)
(ex: ECOR1 contains methylase to methylate Adenine and Cytosine, so enzyme will be blocked by methyl groups)
What are vectors? Which cellular structures are used? What are retroviruses, adenoviruses and herpesviruses used for?
Vectors: Autonomously-replicating DNA used to carry the desired gene to a new cell
(types of vectors: Plasmids, viruses)
Plasmids and viruses can be used (choice depends on organism receiving the gene and size of cloned DNA)
plasmids: Primary vectors in use. Easy to manipulate. However, no “natural” mammalian plasmid replication origins
-Viruses: generally, accept LARGER pieces of foreign DNA. (can be used in mammalian cell lines)
-However inserted DNA must NOT disrupt essential viral genes or increase genome size beyond capsid capacity
retroviruses, adenoviruses, and herpes viruses are used to insert corrective genes into human cells
What are the necessary properties for Vectors?
Necessary Properties for vectors
-Self replication
-Small size is advantageous (less fragile, efficient transformation, easier to purify from chromosomal DNA, more likely to have unique restriction sites)
-Able to avoid destruction by host nucleases (ex: circular)
-Carry a selectable maker gene (ex: antibiotic resistance or auxotrophic marker)
Would marker genes that can be phenotypically screened be useful?
YES, its good to be able to select for something, since you can put a thousand colonies on plate and select for one. With screening you can look at 300 hundred colonies on a plate at a time and will not be able to Screen after transformation occurs. Y
What kind of vector is used for cloning? What are its components ?
A E. coli Plasmid Vector is used for Cloning
The vector (about 2,600 base pairs)
it includes origin of replication (allow to replicate in E.coli, lac Z gene, has ampicillin resistance, and multiple cloying site (stretch of DNA engineered into plasmid containing many restriction sites)
What do shuttle vectors compose of?
Shuttle vectors:
- vectors that can be selected in at least TWO different species (E. coli and another organism)
-Requires two suitable selectable markers and ORIGINS of REPLICATION (E.coli/yeast, E. coli/Mammalian, E. coli/fungi, E. coli/plant, E.coli/other bacteria)
there are NO “natural” mammalian plasmid ORI (origin of replication
Distinguish between vector self-ligation and insert ligation?
In cloning, Vector is cut open with BamHI. Then you try and put in fragment containing Bgl II enzymes.
Take DNA and mix with insert and add ligase in test tube; let incubate and will end up with some parts not ligated, and some insert not inserted anywhere.
Vector that got insert and is ligated together.
Vector self-ligation
Insert ligation:
2 plasmids will confer resistance on antibiotics on E.coli cells : religation or incorporation of insert. Religation occurs more frequently.
REVIEW
Explain the purpose of the blue white screen and how it works
The blue white screen: one molecular technique that allows for the detection of successful ligation of foreign DNA in vector-based cloning
Process:
1. Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasmid has genes for lactose hydrolysis (the lac Z gene encodes the enzyme Beta-galactosidase) and ampicillin resistance
2. Foreign DNA will insert into the lac Z gene . The bacterium receiving the plasmid vector will NOT produce the enzyme Beta-galactosidase if foreign DNA has been inserted into the Plasmid.
3. The recombinant plasmid is introduced to the bacterium , which becomes ampicillin resistant
4. All treated bacteria are spread on a nutrient agar plate containing ampicillin and a Beta-galactosidase substrate and incubated. The Beta-galactosidease substrate is called X-gal
X-gal mimics lactose (lactose that links to dye called Indole(normally colorless) ) if enzyme Beta-galactose cleaves it, cells turn blue
5. The only Bacteria that picked up the plasmid will grow in the presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound.
lac Z cleaves disaccharide of lactose (glucose + galactose)
(if cells express: beta-galactosidase : cells will be blue
-recombinant plasmid will have white cells (since expression is disrupt)
What is PCR and what is useful for?
Polymerase Chain Reaction (PCR)
-enzymatic method to AMPLIFY (make multiple copies) a piece of DNA* to detectable levels
This is useful for
-Cloning a piece of DNA
-Diagnosing genetic diseases (ex: restriction analysis)
-Detecting pathogens
*relatively small specific sequences
What are the three major steps of PCR?
- Denaturation
- Annealing
- Elongation
Describe the process of PCR. Also discuss preparation steps.
PCR (Polymerase Chain Reaction)
Preparation:
1. Add primers, nucleotides and DNA polymerase
First Cycle:
2. DENATURATION: incubate at 94 degrees C for 1 minutL this temperature will separate the strands (double stranded)
3. ANNEALING: incubate at 64 degrees C for 1 minute; this allows primers to attach to single-stranded DNA (stick together)
4. ELONGATION: incubate at 74 degrees C for l minute: DNA polymerase copies the target DNA at this temperature
We must raise it to around 74 degrees because it is the optimal temperature for the polymerase
During each cycle, you make more and more product
What determines the specific times and temperatures chosen for each step in PCR?
- The specific times and temperatures;
94 degrees; temp high enough to denature all DNA and separate strands
choose cool temperature like 64 degrees C to allow primer to hybridize to DNA
Elongation: set to 72 degrees for polymerase - The temperature depends on Length of primer and percentage of GC’s (annealing composition)
-Time chosen based on optimal temperature for Polymerase
-Elongation time depends on the size of product made and
if product is longer and has more nucleotides , you give it more Elongation time
What is unique about the DNA polymerase used in PCR?
it is a THERMAL Polymerase that operates at High temperatures (needed to survive at high temps for denaturation of strands)
What happens during the second cycle of PCR?
The second cycle; repeats the cycle of heating and cooling to make two more copies of target DNA
(other primer anneals to new strand, followed by elongation)
once you reach third cycle, you get product.
What are the functions of thermocycler and Robocycler? How do they differ?
Thermocycler: it will raise and lower temperatures at different tops . The ONE block will heat up and cool down (back and forth)
Robocycler: Has three blocks with set temperatures for denaturation, annealing and elongation
The machine has a robotic arm that can move and pick up all tubes and move them from one block to another during certain times.
- Robocycler WORKS FASTER
How is the amplified product in PCR detected?
Standard PCR: put all reagents in tube, set PCR machine and let run for 30 seconds
-to detect product:
you take sample and run Agarose gel, and use ETHIDIUM BROMIDE (compound that intercalates DNA) and when exposed to UV light will fluoresce.
-
How does real time PCR/Quantitative PCR (qPCR) differ from standard PCR? What are the PCR phases?
Real time PCR/Quantitative PCR: you can Track and see amount of product in every cycle
At 10 molecules, it takes longer to reach Exponential phase
-If have more DNA, reach Exponential phase at an earlier cycle
PCR phases:
-Linear phase : enzyme is not as active
Plateau phase
-Exponential phase
standard PCR: you only see product at end of cycle.
With Quantitative PCR: you can determine how much DNA or RNA you started out with (based on cycle number and when exponential phase occurred)
How do you detect the amount of product produced at each cycle in PCR?
Methods to detect product in cycles of PCR:
1. SYBR: a chemical which intercalates between double stranded DNA to fluoresce (SYBR does NOT fluoresce on its own)
Amount of flourescence correlates to how much product (or Doubles stranded DNA ) is present
- TaqMan (like pac man game) : use Taq polymerase.
- 2 primers ( product you will amplify)
Have a probe that matches sequence in middle of product
probe sits down during polymerization, polymerase as it extends the primer
-probe includes fluorescent molecule and another molecule that quenches fluorescence.
detect extension of polymerase and release fluorescent compound
- disadvantage of Taqman method is you must design specific probe and use quencher and fluorescent molecule for it work; whereas SYBR can use regular primers
