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BIOL 305
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- 1What are the three chemical components that make up DNA?1. Phosphate backbone.
2. Deoxyribose sugars.
3. Nitrogenous bases. - 2What are the two types of DNA bases?1. Purines A and G, which are larger and have two-carbon rings.
2. Pyrimidines C and T, which are smaller and have one-carbon rings. - 3What are Chargaff's Rules?1. The amount of purines is equal to the amount of pyrimidines.
2. The amount of A is equal to G and the amount of C is equal to T. - 4What are complementary bases?Describes the pairing between nitrogenous DNA base types. Due to their shape, purines and pyrimidines form H bonds with each other, three between C and G and two between T and A.
- 5What constitutes a DNA double helix?Two side-by-side strands of assembled nucleotides with complementary base pairs.
- 6Which nucleotide base pair takes more energy to break down?C and G, since they have three H bonds compared to two H bonds between T and A.
- 7Where does the strength in DNA come from?Hydrogen bonds between base pairs.
- 8Which base pair combination is found at higher temperatures and why?G and C, because the increased amount of hydrogen bonds allows the DNA to remain double-stranded at higher temperatures.
- 9What constitutes the backbone of DNA?Alternating phosphates and sugars linked by phosphodiester bonds.
- 10In which direction do sugar-phosphate backbones run and why is this important?They run in opposite, anti-parallel directions, which is important because polymerases read in a 5' to 3' direction.
- 11Would DNA get distorted if T-A replaced A-T or G-C replaced C-G?No, because the nucleotide types take up the same amount of physical space and have the same geometry.
- 12Why does DNA have grooves and what is their purpose?DNA grooves are due to the geometry of nitrogenous base pairs, resulting in major front grooves and minor back grooves. Most proteins have evolved specifically to approach and land on the major front groove.
- 13Who was Rosalind Franklin?A DNA crystallographer whose X-ray image of helical DNA turns allowed Watson and Crick to figure out it groove-based structure.
- 14What are single-nucleotide polymorphisms and why are they important?The most common form of genetic variation, polymerases make about one error per 10^10 bases copied. No errors would mean no variation for natural selection to act upon, and consequently no adaptation to the environment.
- 15How is RNA different from DNA? What is a problem it has?RNA has a U pyrimidine instead of a T. RNA's oxygen is very reactive, so its backbone breaks down easily.
- 16How is the phosphate backbone of DNA formed?Through triphosphates, provide the energy to have themselves added. Two phosphates are cut off and a covalent bond is formed, which results in a phosphate backbone. This is the reason for 5' to 3' directionality, since if an error is made, it is easy to cut off one phosphate.
- 17What does DNA polymerase do and how does it work?DNA polymerase is the primary enzyme which catalyzes the linking of the 3' hydroxyl group of the end nucleotide to the 5' phosphate of nucleotide to be added. It will hold an unpaired base until the correct nucleotide comes in to bind to the active site, then a hydrogen bond will form and the backbone will continue to grow.
- 18What is processivity?Refers to the amount and speed at which nucleotides are added. It is affected by the different levels of proofreading in different enzymes. Highly processive polymerases add hundreds of base pairs per second and stay associated without falling off for thousands of base pairs.
- 19When might low levels of proofreading be beneficial?Viruses need to replicate quickly and with many variations in order to evade immunity, therefore little to no levels of proofreading would be beneficial for them.
- 20Why was RNA sequenced before DNA?1. Easier to get a large amount.
2. Easier to get a pure sample.
2. Less complicated than DNA since it's single-stranded.
3. Shorter than DNA.
4. There was better knowledge of RNA enzymes at the time. - 21How was direct DNA sequencing made possible?1. Relied on previous techniques used in RNA sequencing.
2. Discovered how to purify bacteriophages from DNA genomes. Lambda bacteriophage had overhanging 5’ ends, which meant it was already primed and left scientists with only a few things to sequence. - 22In which direction does proofreading occur?3' to 5', the opposite direction of synthesis.
- 23What were the innovations and limitations of early DNA sequencing?Next step was using oligonucleotides to prime DNA polymerase at any position on a strand. Limitations included sequencing lengths and difficult procedures.
- 24What breakthrough spawned First Generation sequencing technology?The ability to generate large polyacrylamide gels, which provided great resolution.
- 25What is Maxam-Gilbert sequencing?Uses chemicals, not polymerization, to insert breakages at specific bases in a nucleotide sequence.
- 26How does Maxam-Gilbert sequencing work?DNA is radioactively labelled at the 5' end. The sample is split into four separate tubes. Each tube is treated with specific chemicals to break the nucleotide chain at targeted bases. The result is read in the opposite direction of migration, since the smallest DNA pieces will migrate to the bottom the fastest during electrophoresis.
- 27Why is only causing one break ideal for Maxam-Gilbert sequencing?Since only the break closest to the phosphate will be detected.
- 28What is Sanger or dideoxy sequencing?Uses chemical analogues and polymerization.
- 29How does Sanger or dideoxy sequencing work?The primer is radioactively labelled and the DNA sample is split into separate tubes. P32 primer, dNTPs, and DNA polymerase are added. Then, one of four ddNTPs are also added, which results in a truncated DNA fragment.
- 30What does dNTP stand for?Deoxyribonucleotide triphosphate.
- 31What made Sanger or dideoxy sequencing so popular?1. Sequencing accuracy.
2. A robust and easy-to-use method.
3. Possibility of long sequence lengths.
4. Improved detection.
5. Invention of the DNA sequencer or automation.
6. Radioactive labels replaced with fluorescent labels. - 32What is automation or capillary sequencing?Capillary electrophoresis consists of acrylamide in a fine glass capillary. DNA is loaded at the top, and a laser scans and detects it as it moves down the column. The smallest molecules move to the bottom the fastest. More efficient.
- 33What was the first complete genome sequence of a free-living organism and how was it sequenced?H. influenzae, sequenced by shotgun sequencing. Had fewer base pairs and was A-T rich, which meant fewer errors. Fragmented the DNA into small pieces and cloned them, then sequenced from each end. This resulted in a bunch of small sequences which the computer would build up and overlap.
- 34How does shotgun sequencing work?A specific fragment size is selected and litigated into vectors. These are then introduced into an E. coli strain, which is mutated to reduce DNA metabolism. The vectors are then purified and sequenced.
- 35What are the steps of shotgun sequencing?1. Library construction phase, where DNA is isolated, fragmented, and cloned.
2. Random sequencing phase, where DNA fragments are sequenced.
3. Closure phase, where sequences are assembled, edited, annotated, and closed.
4. Genome completion phase, where this is repeated. - 36What are some ways to fix reads that don't overlap during shotgun sequencing?1. Fingerprinting, which makes a DNA oligonucleotide based on a contig and searches fragments for a match.
2. Peptide link, which uses encoding proteins to predict the DNA sequence.
3. Lambda cloning, which carries large inserts to probe for contig ends.
4. PCR, which creates primers from the ends of contigs to amplify DNA. - 37What are some examples of First, Second, and Third Generation sequencing technologies?1. First Generation: Maxam-Gilbert and Sanger.
2. Second Generation: Illumina, Pyrosequencing, and Ion Torrent.
3. Third Generation: Nanoport. - 38What were some of the innovations that lead to sequencing revolutions?1. Microfabrication.
2. Improved image resolution.
3. Biotechnology research and engineering. - 39What is Illumina sequencing?A genome is broken down into double-stranded fragments. Adaptors are added to the ends, which will help add on specific sequences. The DNA is denatured and becomes single-stranded, and is then loaded and attached to a flowcell. The samples bind to primers and PCR amplifies them to make them double-stranded again. These form bridges, then dissociate and form clusters. Each new nucleotide has a fluorescent label so it can be seen as it comes in.
- 40How are Illumina and Sanger sequencing similar?1. Both use capillary gel electrophoresis.
2. Both machines detect fluorescent nucleotide labels. - 41How are Illumina and Sanger sequencing different?1. In Sanger, the fluorescent label is attached to the base itself. In Illumina, the fluorescent label is attached to the O group.
3. Illumina can also continue to sequence the same piece of DNA, which Sanger cannot do. - 42What was one of the innovations of Illumina?Not constraining spots to a grid and instead letting them appear randomly on a flowcell.
- 43What is the difference between Second Generation and Third Generation sequencing?Second Generation sequences by synthesis and uses PCR to make more copies of the gene in question. Third Generation doesn't use a PCR step and instead looks at the original gene in question, and can sequence much longer fragments.
- 44What is the PacBio sequencing method?Real-time sequencing where each well has PCR polymerase tethered to the bottom of a plate, which is see-through and allows a camera to see it from below. This only sees the nucleotide being incorporated due to microfabrication. Fluorescence is attached to phosphate groups.
- 45What is Nanopore sequencing?Sequencers are very small and plug right into computers like a USB. Single-molecule sequencing, so no need for PCR. Long read lengths and can be used for RNA and proteins. Creates a voltage current and has processive activity, which means it can separate two pieces of DNA. The pores slow down the DNA so the computer can keep up. Downside is less accurate.
- 46What is Moore's Law?The power of computer hardware doubles every 2 years and the DNA sequencing capacity doubles every 5 months, therefore the major limitation is lacking the memory or transmission capability to deal with too much data.
- 47What is Ion Torrent?Each well measures when polymerization happens. As hydrogens get cut off, the pH meter will detect the H increase. Error rates can increase with long homo-polymer runs, which is due to the difficulty of telling how many of the same base have been added by the pH increase.
- 48What is the Human Virome Project?A global initiative to identify 99% of all high-consequence viruses in wildlife hosts that are most likely to carry the next pandemic so we can characterize the ones most likely to become zoonotic. $3.4 billion over the next 10 years to sequence every virus that could possibly infect a human.
- 49What is the Human Genome Project?All done through Sanger sequencing with scientists primer walking through the human genome. Was divided into chunks, each was inserted into a bacterial artificial chromosome, and cloned fragments were sequenced in labs around the world. Sequenced 1000 base pairs at a time, very slowly.
- 50What was the race between Celera and the Human Genome Project?Celera directly chopped up entire genome at once and attempted to fuse genome together through overlaps. Human Genome Project created international collaboration and shared their findings publicly as they become available. A public versus private sector race that came down to fundamental ethical questions of who owns a genome.
- 51What was Griffith's experiment?The first experiment suggesting that bacteria are capable of transferring genetic information through transformation. Used two strains of S. pneumoaniae to infect mice, a smooth virulent type and a rough non-virulent type. The smooth strain synthesized a polysaccharide capsule that protected itself from the host's immune system, resulting in the death of the host, while the rough strain did not have the protective capsule and was defeated by the host's immune system. Griffith concluded that the rough strain had been transformed into the lethal smooth strain by a transforming principle that was part of the dead smooth strain bacteria, which we now know was its DNA.
- 52What determines what is the first base pair in a genome diagram?The origin of replication.
- 53Why are bacterial genome diagrams tilted to the left?Bacteria have one single origin of replication. On the opposite end across from this is the terminus, where replication stops. To map E. coli, scientists used to use plasmids to integrate or conjugally transfer, which is why the E. Coli diagram is tilted to the left.
- 54What are all of the enzymes involved in DNA replication?1. Exonuclease acts on the DNA end.
2. Endonucleases are restriction enzymes that cut DNA in the middle of the molecule to create ends.
3. Replication is bi-directional, have leading and lagging strands since polymerases must copy in 5’ to 3’ direction.
4. Helicase wraps itself around and uses energy in the form of ATP to melt DNA into a single strand, which prevents strands from re-annealing and acts as a styrofoam buffer around the DNA backbone.
5. DNA polymerases need a 3’ OH to add nucleotides to. On leading strand, polymerase can keep going. On lagging strand, machinery has to wait for the next stretch of DNA to be melted before the primase can come in and prime the next reverse reaction.
6. RNase H cleaves RNA components.
7. DNA ligase seals nicks on lagging strands. - 55What are the three components of basic viral structure?1. Capsid, part of external coating.
2. Envelope, part of external coating. Absence of an envelope is a naked virus.
3. Virion, a fully-formed virus able to establish an infection in a host cell. - 56Why do viruses have much higher mutation rates?The smaller genomes get, the more mutation rates they have. Also have low levels of proofreading to help replicated quickly and develop immunity.
- 57What is open access publishing?Articles are free to read and free to reuse. Main disadvantage is slow movement of scientific cultural practices. Main advantage is openness helps accelerate scientific advancements and article visibility.
- 58What are some conflicts of interest that have been seen in big publishing journals?1. Generating fake journals for pharmaceutical companies.
2. Investing in global arms trade while publishing medical journals for harm reduction such as Injury Prevention.
3. Publishing publicly funded articles. - 59What are the advantages and disadvantages of impact factors?Impact factors are the frequency with which an average article in a journal has been cited during a particular period. Advantages are not biased by journal size or frequency of issues. Disadvantages are measurement of the journal and not the scientist or quality of the science, too easy a metric for administrators, differs between fields, different calculations pick different winners, and cheaters.
- 60Why are there more genes encoded on leading strands than lagging strands?Higher mutation on the lagging strand. DNA replication system moves faster and gets the right of way, pushes its way through, but at risk of a head-on collision with RNA polymerase, so they move in the same direction.
- 61What are transposons?Genetic elements that can jump and move DNA form one piece to another.
- 62What is a gene copy number?High enzymatic output if a gene is located near the origin, low enzymatic output If a gene is located near the terminus. Genes by the terminus are always the last to be copied.
- 63What is a multi-copy model?E. coli or any fast-growing cell is going to initiate replication multiple times, so multiple origins in comparison to one terminus. Same amount of promoter activity for each, but increased amount of transcripts.
- 64What are some explanations for the percent of transcripts per domain?1. Could be the number of genes exceeds the amount of activators.
2. RNA stability could play a role, mRNA genes around the terminus are more stable than mRNA genes around the origin. This way, the cell can essentially buffer against changes made. - 65What are replisomes?Pol III core, which is three subunits put together, and Pol III holoenzyme, which is three Pol III cores put together, so nine total subunits. A solution to prevent stalling, stopping, and reloading is essentially tethering the polymerases to the DNA. The ring stops a polymerase from diffusing away when it stops and floating away from the cell, this could cost valuable seconds.
- 66What initiates replication and how?DNAa, which is an initiator. Recognizes green binding sites, and though binding, recruits more DNAa to come in and bind. DNA helicase is loaded on and establishes a bubble, then an RNA molecule that can start is added.
- 67What is hemimethylated DNA?Helps time replication and identify and repair mutations. Dam methylase will methylate both strands of a double-stranded DNA molecule, 3’ GATC and 5’ CTAG. SeqA blocks the origin, then will naturally fall off over time. Dam methylase will come in and make the DNA fully methylated, then another round of replication can be initiated.
- 68What was the point of reading the assigned E. Coli paper?Used E. coli to demonstrate that the expression of a reporter gene cassette, comprised by the lac promoter driving expression of gfp, varies depending on its
precise position on the chromosome. Was way higher in some positions, and was completely silenced in others, effectively overriding local lac promoter
control. These effects were not due to differences in gene copy number, but the differences in gene expression.
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- Fichas sobre BIOL 305, creado por Carlie Hinz el 08/10/2018.
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- Carlie Hinz
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