Recombinant DNA Technology Flashcards
Intentionally modifying genomes of organisms for practical purpose
Recombinant DNA technology
3 Goals of recombinant DNA technology
Eliminate
Combine
Create
3 Goals of recombinant DNA technology
-Eliminate
Undesirable Phenotypic traits
3 Goals of recombinant DNA technology
-Combine
Beneficial traits of two or more organisms
3 Goals of recombinant DNA technology
-Create
Organisms that synthesize products human need
Overview of recombinant DNA technology
6
- Isolate plasmid
- Cleave DNA to obtain desired genomes
- Isolate gene
- Insert gene into plasmid
- Insert plasmid (w/ desired gene bacteria)
- Culture bacteria for various use
Physical and chemical agents that produce mutations
Mutagens
Scientists utilize mutagens to
- Create changes in microbes’ genomes to change phenotypes
- Select for and culture cells with beneficial characteristics
____________ alone can be isolated.
Mutated genes
Explain reverse transcriptase
- Isolated from retroviruses (Genome is RNA)
- Uses mRNA template to transcribe molecule of cDNA
- Easier to isolate mRNA molecule for desired protein first
- mRNA of eukaryotes has introns (non-coding regions) removed……. (leaving only coding DNA)
- Allows cloning in prokaryotic cells
Molecules of DNA and RNA produced in cell-free solutions
Synthetic Nucleic Acids
Uses of synthetic nucleic acids
Elucidating the genetic code
Creating genes for specific proteins
Identifying codons for a.a
Elucidating the genetic code
Synthesize a gene for practical use
Creating genes for specific proteins
Synthesizing DNA and RNA “prodes” to locate specific sequences of nucleotides and Synthesizing antisense nucleic acid molecules use
synthetic nucleic acids con’t
Prodes are small sequences of nucleic acid which are labeled with fluorescent marker (enables the identification and presence of specific DNA sequences in certain genes)
Synthesizing DNA and RNA “prodes” to locate specific sequences of nucleotides
Nucleic acid sequence which binds and interferes with genes and/or mRNA to prevent expressions (research conducted to control diseases)
Synthesizing antisense nucleic acid molecules
Bacterial enzymes that cut DNA molecules only at restriction sites (nucleotide palindromes)
Restriction enzymes
Categorized into 2 groups based on type of cut
Sticky ends
Blunt ends
R.E. stands for
Restriction Enzymes
R.E. are named according to
- Genus species
- Order found
- Strain
E.Coli strain R has restriction enzyme
ECoR1
The same____can be used in different organisms
R.E.
After RE cut DNA, ligase can be added to
Ligate the DNA strand
Nucleic acid molecules that deliver a gene into a cell
vectors
What are some useful properties of vectors?
They are small enough to manipulate in a lab
survive inside cells
contain recognizable genetic marker
ensure genetic expression of gene
Vectors include
Viral genosomes transposens and plasmid
Growth on antibodic medium indicates that gene
was delivered succuesfully
A collectin of bacterial or phage clones
Gene libraries
Each clone in library often contains
one gene of an organisms genome
Library may contain all genes of a
single chromosomes
librarys may also contain set of
cDNA complementary to mRNA
Production of a gene library
1- isolate genome (all DNA)
2- cleave genome into fragments using RE
3- insert each fragment into a vector (plasmid)
4- insert each vector into different cells
5- culture cells, then place into tubes (label, store)
Copying (amplifying) large number of identical molecule of DNA molecules
The polymerase chain reaction (PCR)
Critical to amplify DNA in variety of situations
Epidemiologists use to amplify genome of unknown pathogen
-amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores
DNA specific, dNTPs polymerase enzyme, thermocycler
PCR requires
Heat DNA `94 C breaks H-bonds between dsDNA, resulting in 2 separate complementary strands
Denaturation
Mixture is cooled to ~65 C, so primers in a mixture can anneal to a complementary sequence in template strands of DNA
Priming
Temperature is slightly elevated to 72 C and polymerase enzyme synthesizes each template strand of DNA using dNTPs
Extension
PCR can be automated using a _______: machine which heats and col PCR mixture. repeats the 3 steps
Thermocycler
PRC stand for
Polymerase chain reaction
PCR
Hydrogen bonds breaks
Primers anneal to specific DNA sequences
Polymerization occurs, new strands synthesize from ssDNA
PCR DNA is replicated exponentially after
Sever cycles
Gel electrophoresis
Separating DNA Molecules
Explain Gel electrophoresis
Separates molecules based on electrical charge, size, and shape
- Allows scentists to isolate DNA of interest
- Negatively charged DNA drawn toward positive electrode
- Agarose makes up gel; acts as molecular sieve
- Smaller fragments migrate caster then larger ones
- Determines soze by comparing migrated to strandards
Migration of DNA
smallest DNA fragments travel the fastest
DNA transferred from gel to nitrocellulose membrane
Southern blot
Southern blot probes uses to localize ________
DNA sequence of Interest
what uses southern blots
Genetic “fingerprinting”
Diagnosis of infectious disease
Used to detect RNA
Northen Blot
Explain the southen blot technique (4 Steps)
Cut DNA using RE and place cut DNA mixture into separate wells
1) use gel electrophoresis to separate fragments
2) Transfer (blot) DNA fragmens from gel to nitrocellulose membrane
3) Add radioactive probes to membrane
4) incubate and observe for presence of specific DNA sequence
Goal of DNA technology is insertion of DNA into ____
Cell
Uptake of exogenous DNA
Transformation
Viral transfer of DNA from one microbe —>another
Transduction
Transfer to plasmid to another bacteria
Conjugation
Artifical methods are
Electroporation
Protoplast fusion
Injection
Gene gun and microinjection
Injection
Electrical current —-> holes so DNA enters
Electroporation
Expose microbes to polyethylene glycol to promote fusion of 2 microbes (Recombinant DNA)
Protoplast fusion
Beads coats w/ DNA and shot into target tissue
Injection - gene gun and microinjection
Locating genes and specific nucleotide sequences on a nucleic acid molecule
Genetic mapping
DNA microarrays used to screen individuals for inherited disease caused by mutatuins
Genetic screening
Genetic screening can also identify _____________.
Pathogens DNA in blood or tissue
Identifying individuals or organisms by their unique DNA sequence
DNA fignerprinting (pharmaceutical and therapeutic applications)
Pharmaceutical and therapeutic application
Amplify DNA (PCR)
- Cut DNA (using RE)
- -Each individual may have diff. fragment size
- Run DNA on gen electrophoresis
Missing or defective gens replace with notal copies
Gene therapy
With gene therapy some patients immune system reacts
Nehatively
Patient specimens can be examined for presence of gene sequences unique to certain pathogens
Medical diagnosis
Animals cells, tissue, or organs introduced into human body
Xenotransplants
(Agricultrual applications)
Recombinant plants and animals altered by addition of genes from other organisms
Production of transgenic organisms
(agricultrual applications)
Gen from salmonella conveys resistance to gyphosate (round up)
Herbicide resistance
Farmers can kill weeds without killing crops
Agricultrual applications
Herbicide resistance
(Agricultrual applications)
Scientists have removed gene for salt tolerance and inserted into tomato and canola plants
Transgenic plants survive produce fruit and remove salt from soil
salt tolerance
(Agricultrual applications)
crops sprayed with genetically modified bacteria can tolerate mild freezes
Freeze resistance
(Agricultrual applications)
(bt toxin) naturally occurring toxin only harmful to insects
-organic farmers used to reduce insect damage to crops
-gene for Bt toxin inserted into various crop plants
Pest resistance
(Agricultrual applications)
tomatoes allowed to ripen on vine and shelf life increase (gene for enzyme that breaks down pectin suppressed)
Improvements in nutritional value and yield
allows cattle to gain weight more reapidly, have meat with lowe fat contect and produce 10% more milk
BGH