Exam 3 - Part 1 Flashcards
genetic engineering
deliberate modification of organim’s genetic information by directly changing the sequence of nucleic acids in it genome
recombinant DNA technology
procedures used to carry out genetic engineering
biotechnology
use of organisms to form useful products
- grow cells and collect DNA
- use of restriction enzymes and vectors
- put pieces of DNA into vector to put into host and express gene
gel electrophoresis of DNA
- used to separate molecules based on their charge and size
- agrose or acrylamide gels can be used to sparate DNA fragments
- DNA is acidic and it migrates from the negative to the positive end of the gel
- used for separating molecules
what can gel electrophoresis also be used in?
protein analysis
the technique of PCR
- rapid amplification of a speific DNA fragment from a complex mixture of DNA and other cellular components
- pieces of DNA ranging in size from 100 to several thousand base pairs in length can be amplified
- PCR negates the need for some of the steps traditionally required for clining of a gene
what is blotting and what are the three types of blotting techniques?
southern, western, and northern
- gel is fragile, and it can break up, which is why you transfer the gel to paper called nitrocellulose paper
southern blotting technique
used to detect specific DNA fragments
- often uses radioactive DNA hybridization probes
autoradiography
method for detecting radioactively labeled molecules
recombinant DNA cloning vectors
cloning vectors are used to provide many copies of cloned DNA (via replication in a host organism
each type of cloning vector generally has what?
- an origin of replication
- a selectible marker
- a unique restriction site(s) called a multicloning site (MCS) or polylinker
construction of genomic libraries
- used when gene of interest is on a chromosome that has not been fully sequenced
- the library is constructed by cleaving the genome and then cloning the fragments into vectors
- the libraries are screened for the genes of interest in a vareity of ways
what are the most common hosts for recombinant DNA?
- e coli (prokaryotic)
- saccharomyces cerevisiae (eukaroytic host)
how are hosts engineered for recombinant DNA?
- engineered to lack restriction enzymes and RecA
what are the different ways to introduce DNA into microbes?
- transformation
- electroporation
- protoplast fusion
genomic fingerprnting
- also used for microbial classification and determination of phylogenic relationships
- takes advantage of the presence of multi copies of highly conserved and repetitive DNA sequences present in both gram negative an positive bacteria
- restriction enzymes are cleaved and cut into specific fragments and compared
- these sequences are amplified by PCR
- then run on agarose gel and analyzed by a computer
applications of genetic engineering in medicine
- many useful proteins
- gene therapy
applications of genetic engineering in agriculture
- use of genetic engineers allow for the direct transfer of desirable traits to agricultually important animals and plants
industrial fermentation
the mass culture of microbes (or plant and animal cells)
- requires precise control of agitation, temp, pH, and oxygenation
biofuel production
transformation of organic materials into biofuels such as ethanol and hydrogen
what are the types of secondary metabolites?
industrial and agricultural products, food additives, products for human and animal health, biofuels, vaccines
penicillin
requires precise control of nutrients by manipulating C and N source
- this causes the microbe to over produce a secondary metabolite
what is the process of electrophoresis?
- pour agarose into a plate
- put holes in to the top of the plate and load DNA into the wells
- the DNA will travel to the other side of the gel
- the smaller the fragments, the further they will go
why is taq polymerase used in PCR?
- this polymerase is great at withstanding high temperatures, allowing for DNA to be replicated at high temps
western blotting technique
transfer proteins
northern blotting technique
transfer RNA
what is the process of blotting?
- locate fragments
- make probe that is homologous to the DNA to find a specific molecule
- a radioactive label to form a complementary bond
selectible marker
- gene in a vector that allows for an organism to grow in a lab environment with a vector
- can carry antibiotic resistant genes
restriction sites
- restriction enzymes can cut and put in a new gene
- cut out the pieces you want
cosmid
- man-made, is a plamid and virus
what are the different types of vectors?
plasmid, bacteriophage, cosmid, PAC, BAC, YAC
what are some examples of a plasmid vector?
pBR322 and pUC19
PAC
p1 artificial chromosomes
BAC
bacterial artificial chromomes
YAC
yeast artificial chromosomes
what is the process of constructing a recombinant plasmid?
- use restriction enzymes to cut up DNA at specific sites
- recognizes dsDNA and specific pattern
- forms a sticky end (palindrome sequence) for complementary plasmid and the recombinant DNA
- may result in a blunt end where recombinant DNA cannot fit
process of inserting recombinant DNA into host cells
- mimick transformation in the lab
- host does not have restriction enzymes
- has to be competent
competent
- receptive of foreign DNA for transformation
- cold and then heat shock
electroporation
makes DNA more porous, not as widely used
what does RFLP stand for and what is the process?
- restriction fragment length polymerazation
- used in genomic fingerprinting
- cut up with restriction enzymes
- isolate DNA (PCR if needed)
- electrophoresis and blotting
- analyze by looking for highly conserved and repetitive regions
- put into a computer program and compare with other DNA fragments
autonomous replication
- can replicate on its own with the help of an ORI
- independent of host chromosome
interleukins (cytokins)
- proteins produced by the immune system as immunomodulators, communication between immune system components
- used for attacking cancer cells
tumor necrosis factor (TNF)
- types of cytokin
- anti-cancer, destroys cancer cells
metabolic engineering
- genetic manipulation of enzymes
- design new pathways and enzymes involved to make less or more of something
- whole genome shuffeling
what are the products produced during metabolic engineering?
lysine (aa), antibiotics, lipases, lycopene, lactic acid, enzymes
biofuel production
- ethanol made from nonhydrocarbon sources
- agricultural products like corn can be turned into glucose, yeast, and then alcohol
- brazil has the same process with sugar cane
primary metabolites
- minimal reuirements to maintain life, normal growth
- processes like glycolysis, krebs, etc, etc. to make building blocks
secondary metabolites
- enzymes that are not normally made
- molecules not produces under normal conditions
subunit vaccine
pieces of the virus are made in a lab, like a protein or polypeptide, the body thinks you have been infected by the virus and produces antibodies
- uses reverse vaccinology to decide which protein is best to protect the body
annotation
determine location of genes on newly sequenced genome
genome annotation
- locates genes on a genome map
- identifies each ORF in a genome
- uses database to assign function of a gene
paralogs
- two or more genes found alike in the same genome that likely arose from gene duplication
orthologs
two or more genes very similar in different organisms that are predicted to have same functions
proteomics
- study of the proteome, the entire collection of proteins that an organism produces
functional proteomics
information that determines what is actually happening in the cell
metabolomics
identifies all the small molecule metabolites in a cell at a given time
lipidomics
determines a cell’s lipid profile at a given time, allows an assessment of how the environment affects a cell’s membrane
comparative genomic analysis
- has provided info about virulence and evolution as well as potential targets for therapy or vaccines
- for example, comparison of m. tuberculosis with m. leprae has given insight into its evolution as an intracellular parasite
capsid
- protects DNA/RNA
- involves in recognizing the host surface
- facilitates nucleic acid penetration
envelope
composed of host lipid and viral proteins
spike
used for recognition and attachment to host
steps required for replication
- attachment
- entry
- uncoating of genome
- synthesis
- assembly
- release
what does the mechanism of viral replication depend on?
- type of nucleic acid
- naked or enveloped virus
viral routes of entry
respiratory, alimentary, skin, genital, conjunctiva
viral shedding
respiratory or oropharyngeal secretions, feces, skin, urine, milk, genital secretion, blood
viral spread in the body
- Local spread of epithelial surfaces
- Subepithelial invasion/lymphatic spread
- Viremia - spread by the bloodstream
- Invasion of skin, CNS, other organs
- Invasion of the fetus
Factors Contributing to the Viral Pathogenicity: entering the host cell
- Directly via trauma or insect bites
2. Through mucous membrane (respiratory, GI, Urogenital tracts)
lysogens
infected bacterial host
monolayer
- Primary cell cultures
- Fused with myeloma cell
- Results in cell culture formation
- will grow indefinitely
- used to grow viruses
tumor
– growth or lump of tissue;
– benign tumors remain in place
Neoplasia
abnormal new cell growth and reproduction due to loss of regulation
anaplasia
reversion to a more primitive or less differentiated state
metastasis
spread of cancerous cells throughout body
Possible Mechanisms by Which Viruses Cause Cancer
Viral proteins bind host cell tumor suppressor
proteins
• Carry oncogene into cell and insert it into host
genome
• Altered cell regulation
• Insertion of promoter or enhancer next to
cellular oncogene
western blot
extract proteins
northern blot
extract RNA
