Lecture Exam 3 Flashcards
What is recombinant DNA and genetic engineering?
Purposeful manipulation of genetic material; The insertion deletion, or modification of genes to produce organisms with desired traits
What are the goals of recombinant DNA technology?
Make large quantities of the new DNA, make large quantities of the protein encoded by the genes of interest
What materials and techniques are used in recombinant DNA technology?
endonucleases,
What is the function of each material and technique used in recombinant DNA technology?
How do the materials and techniques used in recombinant DNA technology work?
What are some examples of products produced using recombinant DNA techniques?
Foods, antibiotics like penicillin, vitamins, enzymes found in detergents
What is a gene library?
A series of plasmids, each of which contains a portion of DNA. It exists in bacteria that have been transformed with the mixture of DNA, it can be made using complementary DNA which allows us to only utilize functional DNA and we don’t have to worry about introns.
How are gene libraries made?
They are made by cloning the entire DNA or complementary DNA of an organism. First, you cut the DNA of interest with a restriction enzyme, then you cut the DNA of the vector, using the same restriction enzyme to make compatible ends. After that, you can bind the vectors and genes of interest using ligase, this makes recombinant DNA. Transform bacteria with the mixed recombinants and grow them to make a portion of the library. Each gene is copied to a single clone, and all of the clones collectively make up the gene library.
How does one find the clone of interest?
Replica plating first you make a replica of your master plate on nitro cellulose filter. Then, you will treat the filter with detergent to lose the bacteria. Next, you will treat the filter with sodium hydroxide to separate DNA into single strands. Then you will add radioactively labeled probes, the probes will hybridize with the desired gene from the bacterial cells. Next, wash the filter to remove unbound probes, and expose the filter to x-ray film. Lastly, compare the developed film with the replica of the master plate to identify colonies containing the gene of interest.
What is gel electrophoresis?
A physical separation of DNA fragments by size, with the use of agarose gel, and an electrical current, allowing them to be studied further
How does gel electrophoresis work?
DNA is placed in agarose gel and at each end of the plate negative and positive currents are added. This allows the DNA, which has a net negative charge, to move across the agarose gel towards the positive charge. The large DNA fragments will migrate slower than the smaller fragments. You can also add Ethidium bromide, which is an Intercalating agent, to stain the DNA and allows it to glow when exposed to UV light. This allows for visualization of the DNA fragments following gel electrophoresis.
What is southern blotting?
It is utilized after gel electrophoresis as a way to visualize the DNA. DNA bands are transferred to a nitrocellulose filter through blotting.
How is southern blotting done?
The solution passes through the gel and filter to the paper towels. This produces a nitro cellulose filter with DNA fragments positioned exactly is on the gel. The filter is been exposed to a radioactively labeled probe for a specific gene and the Pro bowl, base pair or hybridize with a short sequence present on the gene. The filter is that exposed to x-ray film, and the fragment containing the gene of interest is identified by a band on the developed film.
What are some of the uses of southern blotting?
DNA fingerprinting,
What is PCR?
It is the production of a large number of identical molecules of DNA in vitro
How does PCR work?
It is a repetitive process consisting of three steps. First, denaturation using heat at 94 C for 1 min, second priming (dNTPs and Taq DNA polymerase) or annealing (stick to complementary DNA) third extension. This process can be automated by using a thermocycler. They will replicate exponentially.
What are some of the uses of PCR?
Epidemiologists use PCR to amplify the genome of an unknown pathogen, for example west Nile virus. It is also used to analyze, ancient DNA, for example, from Neanderthals. It is used forensically to amplify DNA found in blood semen or tissue samples. It is used to determine paternity from tissue or blood samples between two individuals believed to be related.
What is RT PCR?
Reverse transcriptase polymerase chain reaction. It is taking a sample of RNA and using that to make a complementary DNA and then using the complementary DNA to amplify through PCR.
Why would you use RT PCR?
This is used with viruses that have RNA and not DNA, monitor gene expression, diagnose infection, genetic screening
What are DNA microarrays?
Using RT PCR to build a gene test to show which genes are active and inactive.
How are DNA microarrays made?
Probes are prepared by taking complementary DNA that is normal and complementary DNA that is not for example, from a tumor then reverse transcriptase PCR is done, and each type of complementary DNA is labeled with fluorescent dye. You then combine equal amounts of the normal and tumor, complementary, DNA, and then you hybridize probe to the MicroArray and scan it. The ones that are normal are green. The ones that are from the tumor are red and commonalities between the normal and tumor show up as yellow. This is been studied and compared to understand what the genetic make up is of the tumor and how to best treat it.
What are some of the uses of microarrays?
Monitor gene expression, diagnose disease specifically, genetic screening
How is DNA sequenced?
Why sequence DNA?
What are ways that DNA can be transferred from Cell to cell?
How do the transfer methods from Cell to Cell work?
What is CRISPR?
Clustered regularly interspaced short palindromic repeats. It is a mechanism for bacterial immunity. They are similar to what we find with restriction enzymes.
How does CRISPR work?
When aphasia injects its DNA, the CRISPR associated proteins snip sections of the phage DNA and insert them into the crisper array as new spacers. The spacers act like wanted posters and help the cell recognize potential bacteriophages in the future. This can be considered the immunological memory for this bacteria cell and its descendants. If a phage tries to infect a cell in the future, and there is CRISPR sequence available for that particular phage, it will form CRISPR RNA, and the CRISPR RNA guides molecular machinery to target and destroy the viral genome.
Cas 9 and the guide RNA targets the desired sequence and breaks the double stranded DNA. This is been repaired by the nonhomologous end joining mechanism. This will result in insertions and deletions (these are frameshifts: defective or truncated proteins).
What can CRISPR be used for?
It was figured out that this could be utilized to knock out certain undesirable genes. If utilized with humans, this would allow for modifying genetic code before offspring are born.
Why was there a moratorium on recombinant DNA experiments from 1974 to 1981?
There was a lot of fear surrounding the use of recombinant. They were seen to be dangerous and unnatural people were worried that they could escape and wreak havoc on the general populace. There was also the question of bioterrorism, and the potential for making new pathogens.
Why was the moratorium lifted?
Recombinance could not be shown to cause illness, E. coli strains that are used cannot infect humans, recombinant E. coli with mammalian genes were observed in nature and were out competed by wild type. E. coli, recombinant E. coli could be controlled by standard sanitary practices.
What are some modern day concerns about recumbent DNA technology?
The ethics of national DNA databases and the ethics of super babies or designer genetic babies
What are some of the benefits of recombinant DNA technology?
Crop plants that make useful foreign proteins, such as golden rice. Medicines for example, insulin Humira and human growth hormone. Nano technology.
What are some of the risks of recombinant DNA technology?
Bioterrorism, ethical implications of national DNA, databases and privacy, ethical implications of super babies and genetic designer babies
What is automated nucleotide sequencing?
It is DNA sequencing using fluorescent deoxynucleotides, or chain terminators. DNA is passed through gel electrophoresis, and colored with fluorescent deoxy nucleotides. As it fully passes through the gel, it is red by an automated electronic eye which records the information and allows for DNA sequencing to occur.
What is a nucleotide sequencing?
It is the determination of the order of bases in a piece of DNA
What are some uses of a nucleotide sequencing?
The human genome project, taxonomy, personalized medicine
What are viruses?
What is their basic structure?
How were viruses discovered?
What does it mean to be an obligate intracellular parasite?
Why are viruses and prions obligate
intracellular parasites?
What problems do all obligate intracellular parasites share?
1) making contact with a suitable host;
2) gaining entrance to the host’s body (portals of entry);
3) getting to the right host cell/tissue; and 4) gaining entry to the right host cells
How do viruses solve their problems?
What is an envelope?
What is the function of an envelope?
Of what is an envelope it made?
A portion of cell membrane stolen from host cell during budding.
What sorts of viruses have envelopes?
From what cell component(s) do viruses get their envelopes?
What is a capsid?
What is the function of a capsid?
Of what is it made?
What is host range and what determines host range?
It is the spectrum of hosts that a given virus can infect.
What is host cell specificity and what determines host cell specificity?
It means that the virus can only infect certain types of cells (tissue or cell specificity), which are found only in certain types of hosts (humans vs. dogs). Specificity is determined by ligands or adhesins on virus fitting with receptors on cell.
How did viruses originate?
a) co-evolution of viruses and cells (both had independent origins);
b) evolution from plasmids or transposons (retroviruses = fancy transposons);
c) viruses are degenerate cells
What is mimivirus and what does it suggest about viral origins?
What are some of the ways that viruses can be classified?
viruses classified by type and structure of nucleic acids method of replication, host range, etc.
what are the bases of viral
taxonomy?
What is a bacteriophage?
A type of virus that inserts its genome into a bacterial cell to replicate its genome.
What is lytic replication?
This is the use of cell machinery to make new virus particles, which are
What are the stages of lytic replication?
Attachment (phage binds onto the bacterial cell via tail fibers), penetration/entry (phage injects its genome into cell) synthesis/biosynthesis (causes the host genome to be fragmented, giving the virus genome priority in synthesis), maturation/assembly (capsid proteins are assembled into phages), release (lysis of cell, phages will then go infect other bacteria)
What is a temperate phage
Healthy bacteria that could be utilized in a way that allows the phage genome to be taken up along with the cell’s genome and allow the cell to continue living while the phage is being served.
What is lysogenic replication?
It is when a phage inserts its genome into a host cell and uses the mechanics of the cell to make new phages. It ends in cell lysis.
What is lysogeny?
This is when the host cell does not die, it follows lytic replication except it integrates into the host genome. The progeny of this cell will now include the phage genome alongside the bacterial DNA. Once the cell starts to die, then it will enter into the lytic cycle and end in cell lysis.
What is a lysogen?
A bacterial cell or strain that has been infected with a temperate virus, one that does not cause destruction of the cell.
What is a prophage?
Prophage is a bacterial cell that has phage genome in it that has combined with the bacterial cell, it is the progeny of the lysogen.
How does lysogenic replication differ from lytic replication?
Lysogenic replication combines bacterial DNA with phage genome and cell progeny have both phage and bacterial genome. Lytic replication is complete cell takeover and DNA is rendered inactive and phage DNA is exclusively made.
How do enveloped viruses replicate?
They attach to a cell via ligands/adhesins, use cell machinery to replicate genome, new viruses exit cell via lysis or budding.
What are the steps in enveloped virus replication?
- Attachment – virus uses ligand to latch on to host receptor
- Penetration/Entry – virus envelope fuses with cell membrane (this is ONLY done by enveloped virus)
- Uncoating – virus capsid is lysed, releasing viral genome
- Synthesis/Biosynthesis – virus hijacks cellular machinery to make new virus components
- Maturation/Assembly – virus
components self-assemble - Release – new viruses may be released into the environment via lysis or budding
What is budding?
What are some examples of antiviral drugs and how do they work?
Neuraminidase inhibitor = oseltamavir (Tamiflu®),
uncoating inhibitor = rimantidine (Flumadine®)
protease inhibitors (Indinavir®);
fusion inhibitors (Fuzeon®);
integrase inhibitors (Isentress®);
second-receptor inhibitor (Maraviroc®)
What are latent viral infections?
They are viruses that the body is not able to completely clear, and they will go through “outbreak” cycles that span years.
What types of viruses can set up latent infections?
EBV, HSVI, HSVII, VZV
What is a provirus?
A form of a virus that is integrated into the genetic material of a host cell and by replicating with it can be transmitted from one cell generation to the next without causing lysis
What is teratogenesis?
Something that induces defects during embryogenesis
What are some examples of teratogenic viruses?
Rubella, cytomegalovirus (CMV), HSV I and II.
How can viruses cause teratogenesis?
What is oncogenesis/neoplasia?
oncogenesis is malignant growth
neoplasia is uncontrolled growth
How is cell growth regulated?
What are the mechanisms of viral
oncogenesis?
What are some examples of oncogenic viruses?
What sort of virus is influenza?
RNA Virus
What is the structure of influenza?
8 separate RNA segments in helical structure, it is enveloped (stolen host cell membrane) studded with protein spikes (Hemaggutinin and Neuraminidase)
What are H and N antigens of influenza?
Antigen (antibody generating) Hemaggutinin and Neuraminidase, these are the protein spikes on influenza. Human viruses use H1 H2 H3 and N1 and N2.
What are the functions of the H and N antigens?
They are the ligands that influeza uses to bind to cell’s ligand receptors, sialic acid (found in cells w/in respiratory tract) (ex: Flu H1N1)
What is antigenic drift, and how does it occur?
Errors when the virus genome segments are being replicated (RNA polymerase is not high fidelity-proofreading is poor). The changed virus looks new to the immune system.
What practices, typically seen in Asia, foster antigenic shift?
Wet markets, where animals are kept alive in the marketplace in close contact with other animals and humans. This allows for easy transmission of pathogens and zoonoses.
How are viruses cultured?
Whole living beings, bacteria, (bacteriophage), Plants and animals (embryonated chicken eggs), cell tissue (living human tissue in labs)
What is cytopathic effect?
The effect that certain viruses have on cells. Example: uninfected cells vs. cells infected with measles virus look different.
What is cellular transformation?
describes the changes associated with loss of normal homeostatic control, particularly of cell division.
What are prions?
Misfolded proteins, they have undergone conformational change.
