Chapter 9: Biotechnology and Recombinant DNA
Restriction Enzymes
(Type II/useful for rDNA technology) -Cut DNA at defined positions close to or within recognition sequences -Typically 4-, 6-, or 8- base sequences (cutting frequency) *6-,8- base sequences more useful,* *base pair cutters = 4 to the whatever power (base sequence* -Cut sequences the same way each time -Some produce blunt ends, others produce staggered (sticky) ends. -Staggered ends can be used to join two pieces of DNA with complementary ends *after you cut the particular enzyme, you will get sticky ends*
How is the amplified product detected? How does real time PCR/quantitative PCR (qPCR) differ from standard PCR? What is RT-PCR?
*Amplified product is detected through gel electrophoresis* *Real Time PCR (aka Quantative PCR)* -use a fluorescent dye *can see the amount of product you have at each cycle and can graph it* -useful for determining how much starting product you have *RT-PCR = REVERSE TRANSCRIPTASE PCR *(BE CARE WITH THE ABBREVIATIONS)) for *real time PCR, he will always write it out or say quantitative PCR*
cDNA Library Construction:
*Complementary DNA (cDNA) made from mRNA by reverse transcriptase* *cDNAs then cloned into plasmid or viral vector* -Common for eukaryotic libraries *long mRNAs may not be completely reverse transcribed into DNA* -almost always used for eukaryotes -use a primer that is a long stretch of T'S poly T tail -reverse PCR *will not have any introns, will only have genes encoding for proteins and will get coded
Microinjection
-injecting DNA directly into the cell -impractical for bacterial and fungal cells because the cells are too small -uses a glass micropipette to inject DNA into the cell
*List some ways to insert foreign DNA into cells*
1.) *electroporation* 2.) *chemical transformation* 3.) *protoplast fusion* 4.) *gene gun* 5.) *microinjection*
Gene Therapy
One approach: *replace or augment faulty gene w/normal one* -relies on finding a delivery system to carry correct gene to affected cells. -Gene must be delivered inside target cells and work properly for long term w/out causing adverse effects. -Although most gene deliveries performed by adeno- and retroviruses, plasmid vectors and other strategies being tried -Although some success reported, few patients have received effective treatments and much research still required. Second Approach: *inhibit the expression of defective gene by the following* - RNA interference (RNAi): a eukaryotic biological process in which RNA molecules inhibit gene expression * two small RNA molecules are central to RNAi* - microRNA (miRNA) - small/short interfering RNA (siRNA) *KNOW DIFFERENCE*
What can you use to obtain a genomic or cDNA clone?
PCR, it allows for amplification
Chemical Transformation
*For E. coli (bacteria)* 1.) cells are incubated with DNA in ice cold calcium chloride 2.) then given a mild heat shock* For yeast 1.) cells in incubated with DNA in ice cold lithium chloride 2.) then given a mild heat shock *positively charged ions + negatively charged DNA attract* *Heat shock (42°C) thought to work by transiently opening gated membrane channels*
Genomic Library Construction:
*GOAL OF GENOMIC LIBRARY* --> *make collection of clones large enough to ensure that at least 1 clone exists for every gene in organism* -make large enough to ensure you have all pieces What library size (# genome equivalents) is necessary to ensure that all sequences of the genome are represented? -5 genome equivalents necessary to ensure that all genomic sequences will be represented w/95% probability -10 genome equivalents --> gives 99% probability Choice of RESTRICTION NUCLEASE:? -choose a 4 base pair cutter and do only partial digestion so you have random distribution of fragments
*Recombinant DNA*
*Recombinant DNA (rDNA) technology/genetic engineering* -procedures that are used to join together (recombine) DNA segments in vitro
Nanotechnology
-Making tiny tiny robots/molecules and injecting them into your body and having them fix the problem and then taking them out -drug delivery -Involved with the design and manufacture of electronic circuits and devices built at the molecular level (the object has one dimension that is in the order of a nanometer) -Bacteria can make molecule-sized particles that could provide the needed small wires and components -Bacteria are being used to produce nano-spheres for drug targeting and delivery
Reverse Transcription
-Reverse Transcriptase *converts RNA into DNA* -first cycle is a constant temperature for a certain amount of time Central Dogma: DNA - RNA - PROTEIN (so backwards)
Polymerase Chain Reaction (PCR)
-method of amplifying DNA to a detectable amount -useful for the following: 1.) Cloning a piece of DNA 2.) Sequencing DNA 3.) Diagnosing genetic diseases (ex: restriction analysis) 4.) Detecting pathogens
Tools and Techniques of Recombinant DNA technology
-restriction enzymes -vectors
Safety Issues and Ethics of using rDNA *Will definitely ask a question on this*
-will always be concerns regarding safety of any new technology, genetic modification and biotechnology - virtually impossible to prove something is entirely safe under all conceivable conditions -Organisms in lab modified to avoid accidental release (genes deleted or suicide genes added) -Genetically modified crops must be safe for consumption and for environment (herbicide resistant crop plants could pollinate related weed species) -If genetic screening for disease becomes routine, who will have access to an individual's genetic information? -*Bacillus thuringiensis*
What is a clone?
1) Population of cells arising from a single parent cell 2) Processes used to create copies of DNA fragments a colony is a clonal population -ex: if you cloned a gene, you're just making more copies of that gene
PCR Steps:
1.) Denaturation *94 degrees C @ 1 min* -Incubate: 1.) target DNA 2.) primers 3.) deoxynucleotides 4.) DNA polymerase *separation of the DNA strands* 2.) Annealing *60 degrees C @ 1 min* -Primers attach to single stranded DNA during incubation 3.) Elongation *72 degrees C @ 1 min* -2 copies of target DNA are formed Robocycler Thermocycler
List the Phases of PCR
1.) Exponential 2.) Linear 3.) Plateau
What Cell Types are used to make Gene Products?
Bacteria (E. coli) -easily grown -can carry plasmids -genomics are known (i.e. inducible promoters) Disadvantages: - Need to eliminate endotoxin from products - Does not normally secrete products (must lyse cells to to get product)
Forward Genetics vs Reverse Genetics
Forward genetics ("classical genetics"): -starts with a phenotype and identifies the responsible gene Reverse genetics: -starts with a particular gene and assays the effect of its disruption
How do you sequence an organism's genome?
Random Shotgun Sequencing 1.) Isolate the DNA 2.) Fragment DNA with restriction enzymes 3.) Clone DNA in a bacterial artificial chromosome 4.) Sequence DNA fragments 5.) Assemble Sequences 6.) Edit sequences, fill in gaps *gap sequences are determined by colony hybridization*
Other Cell Types used to make Gene Products
*Saccharomyces cerevisiae* -easily grown -can carry plasmids -genomics are known -Greater chance of expressing eukaryotic genes -More likely to correctly modify eukaryotic proteins than bacteria -Likely to continuously secrete products Plant cells and whole plants -May express eukaryotic genes easily -Low risk of product contamination by mammalian pathogens -Large scale low-cost production Mammalian cells -May express eukaryotic genes easily -Well suited to make proteins for medical use -secreted and low risk of toxins or allergens -Harder to grow
What is a DNA library ?
*a collection of DNA fragments of one organism, each carried by a plasmid or virus and cloned in an appropriate host.* *a collection of DNA fragments that have been cloned into vectors (plasmids) so researchers can identify & isolate DNA fragments that interest them for further study* --> GOOGLE
what are vectors?
*autonomously replicating DNA used to carry desired gene to a new cell* -Plasmids & viruses can be used. (choice depends on organism receiving the gene and size of cloned DNA) -PLASMIDS *most commonly used because easy to manipulate* -VIRUSES *accept larger pieces of foreign DNA* *retroviruses, adenoviruses and herpes viruses used to insert corrective genes into human cells.* No "natural" mammalian ORIs
Genomic library vs Cdna library
*cDNA clone will contain only sequences found in the mRNA* *the genomic clone could have the sequences of the entire gene* *cDNA library will not contain a clone of every gene of the organism* *genomic library could have a clone of every gene for that organism*
What is RNAi also called?
*gene silencing* UNDERSTAND DIFFERNCE BETWEEN using micro RNA'S & small interfering RNAs --> Revisit Cell Biology Notes
Biotechnology
*manipulation of living organisms, or cell components to produce useful products* ex: food, antibiotics
What are shuttle vectors?
*vectors that can replicate in at least 2 different species* *require suitable selectable markers and origins of replication* ex: -(E.coli/yeast -E.coli "mammalian" -E.coli fungi -E. coli/plant -E.coli/other bacteria
The Blue White Screen
-*A molecular technique that allows for the detection of successful ligations in vector based cloning* *screening technique that allows for rapid, convenient detection of recombinant bacteria in vector-based molecular cloning experiments. 1.) Plasmid DNA + foreign DNA --> both cut with same restriction enzymes (NOTE: Plasmid has 2 genes) a.) B. galactosidase gene (lactose hydrolysis) b.) gene for ampicillin resistance 2.) Foreign DNA inserted *into B. gal gene* --> bacteria that receive this new plasmid vector WILL NOT produce B.gal. (because foreign DNA has been inserted into the plasmid) 3.) Recombinant plasmid inserted into bacteria --> bacteria becomes *ampicillin resistant* (because it took up the foreign DNA so it will not produce the B. gal gene) 4.) All treated bacteria spread on nutrient agar plate w/ampicillin and B. gal substrate ( 5.) Bacteria that picked up plasmid with foreign DNA grow in presence of ampicillin (they took up the ampicillin resistance gene) + will produce white colonies. -bacteria that hydrolyze X-gal --> produce galactose + indigo which turns the colonies blue. *bacteria that acquired new trait --> white colonies* bacteria that kept old trait --> blue colonies
Synthetic DNA
-DNA can be made chemically on a solid matrix using a DNA synthesis machine -2 complementary single stranded DNA molecules can be made and then hybridized to generate a double stranded DNA molecule Limitations: - less than 200 bases in length - gene sequence will be ambiguous when only protein sequence is available -although fragments can be ligated together, not typically used for gene cloning - more commonly used for primers and probes Synthetic DNA -primers are used by polymerases to initiate replication -probes are used when you do hybridization and to allneal sequences -can use synthetic DNA to make any type of primer of for the Taqman method
Name and Describe the 2 types of DNA libraries
-Genomic library: contains DNA fragments representing the entire genome of an organism -cDNA library: contains only complementary DNA molecules synthesized from mRNA molecules in a cell
Why do bacteria produce restriction enzymes? What is their purpose?
-RESTRICT the ability of foreign DNA (ex: bacteriophage DNA) to infect/invade the host bacterial cell by cleaving it How do host bacterial cells protect themselves from getting cut by restriction enzymes? -The host DNA is MODIFIED by methylation of their sequences at C or A nucleotides -This modification protects bacterial host DNA from degradation by its own restriction enzyme -Called *restriction modification system*
Forensic Microbiology
-Relatively new specialty of microbiology -The study of microbes to to determine the path of an outbreak, the identity of a criminal or the origin of a particular strain of contagion or biological weapon. -microbes as terrorist weapons (i.e. anthrax enclosed in envelopes to sicken or kill victims) -Microbes as a factor in cases of medical negligence -The deliberate infection of people with a communicable disease -Intentional food contamination. -Must be done in a precise, methodical manner that allows courts to draw conclusions from the data
Protoplast Fusion
-Takes advantage of the fusion properties of protoplasts: fusion rate increases in the presence of PEG -most valuable in plant and algal cells 1.) Bacterial cell walls enzymatically digested --> producing protoplasts 2.) protoplasts treated w/polyethylene glycol in solution 3.) Protoplasts fuse 4.) Segments of the 2 chromosomes recombine 5.) Recombinant cell grows new cell wall
The Taqman Method
-TaqMan probes are hydrolysis probes designed to increase specificity of quantitative PCR. -taqman probe has a fluorphore on it, and as its going, its lighting up, follows PCR to make the experiment is happening *allows you to figure out how much product you have* -each round you release more and more fluorescence *way of quantifiying how much DNA you have made*
Argobacterium tumefaciences and Crown Gall Disease
-bacterium A. tumefaciens infects specific plants at wound sites -bacteria contains naturally occurring plasmid (Ti) -part of Ti plasmid, called T DNA, integrates into genome of infected plant. -T DNA contains genes for opine synthesis and tumor production (crown gall). -Opine = unique amino acid used by bacteria as carbon and nitrogen sources
Electroporation
-making pores in cell using short, but controlled electrical pulses -forces DNA into cell -A controlled short (millisecond) but powerful electrical pulse induces temporary hydrophilic pores in the cell membrane; DNA can then enter the cell -Generally applicable to all cells -*some organisms with cell walls may require prior conversion to protoplasts* (plant cells and algae?) (Not necessary for bacteria or yeast)
Colony Hybridization
-master plate has everything in it (colonies of bacteria containing cloned segments of foreign DNA) 1.) make replica of master plate on nitrocellulose filter (paper) 2.) treat filer w/detergent --> lyses bacteria 3.) treat filter w/NaOH--> separates DNA into single strands 4.) add radioactively labeled probes 5.) probe will hybridize w/desired gene from bacterial cells 6.) wash filter to remove unbound probe and expose filter to X-ray film 7.) compare developed film w/master plate to identify colonies containing genes of interest
Accomplishments using Agrobacterium to insert rDNA into plants
-plants resistant to herbicide glyphosphate - herbicide inhibits enzyme responsible for synthesizing aromatic amino acids - mutant gene resistant to herbicide selected in Salmonella bacteria then introduced into crop plants -Bt toxin gene introduced into a number of plants (i.e. cotton and potatoes) -insects that eat plants are killed
Gene Gun/Biolistic Transformation
-small speck of gold or tungsten is coated with DNA and propelled into the cell -Once particle gets into cell, if it lands on nucleus, genes may come off and may be incorporated into nucleus Used predominately for: 1.)plant cells 2.)C. elegans cells 3.)yeast mitochondria. Other applications include insect, animal and human cells -Burst of helium
Bioinformatics
-the science of understanding the function of genes through computer assisted analysis -DNA sequences are stored in a web based database known as GenBank (provided by the National Center for Biotechnology Information (NCBI). -Genomic information can be searched with computer programs to find: - specific sequences (i.e. motifs) - similar sequences in other organisms - ORFs -Proteomics- is the sciences of determining all the proteins expressed in a cell
How are clones made?
1.) Isolate vector ex: plasmid 2.) Cleave your DNA into fragments using enzymes 3.) Insert gene/DNA into plasmid 4.) plasmid taken up by bacterium 5.) cells with gene of interest are cloned 6.) make your product/copies
what are ideal properties for vectors to have?
1.) Must be able to self-replicate 2.) Must be small size that facilitates manipulation outside the cell 3.) Must be able to avoid destruction by host nucleases (i.e. circular) 4.) Must carry a selectable marker gene (i.e antibiotic resistance or auxotrophic marker)
cDNA Library Construction Steps
1.) gene composed of exons and introns is transcribed to RNA by RNA polymerase 2.) processing enzymes in nucleus remove intron-derived RNA and splice together the exon derived RNA into mRNA 3.) isolate mRNA from cell and add reverse transcriptase 4.) first strand of DNA is made 5.) mRNA is digested by reverse transcriptase 6.) add DNA polymerase to make second strand of DNA
DNA Vaccine (a therapeutic application of rDNA)
1.) gene for an immunogen 2.) insert gene into an expression plasmid 3.) transform bacterial cells, grow bacteria, purify plasmid DNA 4.) immunize with immunogen expressing plasmid
What can clones be used for?
1.) make copies of gene 2.) make protein product of a gene
Using the Ti plasmid as a vector for genetic modification in plants
1.) plasmid removed from bacterium and T-DNA is cut by restriction enzyme 2.) Foreign DNA cut by same enzyme 3.) foreign DNA inserted into T-DNA of plasmid 4.) plasmid reinserted into agrobacterium 5.) The bacterium used to insert T-DNA carrying foreign gene into chromosome of plant cell 6.) The plant cells grown in culture 7.) plant generated from cell clone. All of its cells carry foreign gene and may express it as a new cell.
What are some examples of other agricultural traits?
1.) resistance to other herbicides 2.) male sterility 3.) flower color 4.) modified fatty acids 5.) virus resistance
How do enzymes cut?
1.) restriction enzyme cuts double stranded DNA at particular recognition site 2.) cuts produce a DNA fragment with two sticky (staggered) ends -DNA from another source (perhaps plasmid) cut with same restriction enzyme- 3.) When 2 such fragments of DNA cut by same restriction enzyme come together, they can join by base pairing (THEY ARE COMPLEMENTARY) 4.) joined fragments usually form a linear molecule or a circular molecule. Other combinations of fragments can also occur. 5.) DNA ligase used to unite backbones of the 2 DNA fragments, producing a molecule of recombinant DNA *must have the same restriction enzyme in order for them to stick together*
Human Genome Project
GOAL: -sequence the 3 billion base pair human genome in 15 years DNA -helped by random shotgun sequencing and sequecing advances -complete in 2003 RESULTS: -less than 2% encodes product (remainder is junk DNA) -human genome can now be sequenced for $1,000 a day if you have a cluster of HiSeq X ten sequencing machine (costs 10 million dollars)
What are some therapeutic Applications of rDNA?
Human enzymes and other proteins - human insulin - human hormone somatostatin (5 mg = 50,000 sheep brains or 8 L bacterial culture) Subunit vaccines - specific protein from a pathogen (purified from yeast or expressed as a viral surface protein) DNA vaccines - injection of bacterial plasmids carrying genes for pathogens antigens. -The expressed protein then produces an immunological response Gene therapy - replace defective or missing genes
Natural vs Artificial Selection
Natural selection: *organisms with characteristics that enhance survival are more likely to survive in nature*. -examples: -peacock feathers (male has brighter feather to attract a female) Artificial selection: *humans select desirable breeds of animals or strains of plants* -examples: - a farmer chooses high milk producing cows for breeding
Mutagens
Random mutagenesis: *exposure to mutagen* -can be used to increase the chances of obtaining a desired strain -screening for mutants -ex: Radiating fungus generated a strain of fungus that produced 1000x penicillin Site directed mutagenesis: *mutation created at a defined site in a DNA molecule -can be used to make a specific change in a gene
