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MMBIO 240 Unit 1
| Question | Answer |
|---|---|
| Nucleotide | Nitrogenous base, ribose sugar, triphosphate. |
| What is each carbon in DNA/RNA connected to? | C1: Nitrogenous base C2: RNA- OH, DNA-H C3: -OH C4: -H C5: Phosphate group |
| The bases of nucleotides | Pyrimidine: 5 carbon sugar- Thymine/Uracil, Cytosine Purine: 5 c sugar fused to a 6 c sugar- Adenine, Guanine |
| Defining characteristics of each nitrogenous base | Adenine- missing an oxygen, has NH2 Guanine- has oxygen Cytosine- missing O2, has NH2 Thymine- 2 oxygens Uracil- methyl group |
| Nucleoslide | Base and sugar |
| Single letter code meanings for N, Y, R | N- Any of the nitrogenous bases Y- Any pyrimidine R- Any Purine |
| Why is DNA structured the way it is? (hint: why base and backbone characteristics) | The bases are hydrophobic, the backbone is hydrophilic, allowing new nucleotides to be added from 5' -->3' |
| 4 differences between DNA and RNA | 1. Deoxyribonucleic acid lacks O2 on C2 2. RNA uses Uracil instead of Thymine 3. DNA usually double stranded, RNA usually single stranded 4. DNA is blueprint/code, RNA is a copy that actually does something |
| How new nucleotides are added | 5'-->3' When the adenosine triphosphate (or other nucleotide) is bound to the backbone, it's a dehydration reaction where the phosphate and hydroxyl group bind, leaving an oxygen and releasing the 2 other phosphate groups. |
| Polymerization | Polymerase adding new nucleotides based off of the template strand. |
| Discovery of the structure of DNA | Franklin's x-ray crystallography- double helix double stranded Watson and Crick- discovered pairings and location of components |
| Watson and Crick model of DNA | 2 antiparallel strands (5' lines up w 3' of other), outer has sugar-phosphate backbone, inside purines paired with pyrimidines A:T with 2 H bonds, C:G with 3 H bonds. Major and minor groove because of how it assembles. |
| Sense/Antisense strand | The sense strand is the gene itself. To get that gene, new DNA/RNA is synthesized using the ANTISENSE strand so that the sequence ends up being the SAME as the sense strand. Applies to regions of DNA, so sometimes sense is top, sometimes bottom. |
| Denaturation of DNA | dsDNA-->ssDNA through: 1. Heat 2. pH 3. Chemicals 4. Enzymes (proteins break bonds) |
| Conventional rules of labeling DNA | Top left 5', bottom left '3, top sense, bottom antisense. |
| Central dogma | DNA polymerase replicates DNA RNA polymerase transcribes DNA to mRNA (or others) Ribosomes translate RNA to protein |
| Exceptions to central dogma | 1. Self-replicating proteins: prions 2. Reverse transcriptase taking RNA-->DNA 3. Non-coding RNA's 4. Self-replicating RNAs in viruses |
| Griffith's Transformation experiment | Before: traits passed down, likely amino acids Injected living, dead, and living R cell+dead S cell which killed the mouse because genetic info transferred. Conclusions: Genetic info can transfer and give new traits. But WHAT is being transferred? |
| Avery, MacLeod, McCarthy experiment | Transformation with cultured S/R cells, Centrifuge to blend. DNA not PURE so not conclusive. Destroy protein: transforms. Destroy RNA: transforms. Destroy DNA: NO transform. Conclusion: Nucleic acid likely genetic material, not believed cuz only 4 bases. |
| Tetranucleotide structure | All 4 nucleotides in one structure proposed by Levene. Equal concentration of nucleotides. WRONG! |
| Chargaff experiment | Base content NOT even from different sources. Concluded: -Concentration of A=T -Concentration of C=G -Disproved tetranucleotide structure |
| Hershey and Chase experiment | Blender. Labeled DNA with p32 (radioactive phosphorous), label protein with s35 (radioactive sulfur). Phage injects genetic material for replication, blend up cell, left is genetic material: DNA. |
| The process of translation | Hydroxyl group comes off of carboxyl group of C-terminus end of one protein, one hydrogen comes off of amino group of N-terminus end of another protein. Dehydration reaction taking out H2O, makes covalent bond. |
| How to measure protein concentration | By sending light of 280 nm through sample, because tyrosine and tryptophan absorb light at 280 nm it shows concentration. |
| Importance of methonine | Coded by AUG, it is the start of every amino acid sequence. Can be cut off later. Sulfur allows for radioactive labeling. |
| Importance of Cystine | Only amino acid that can covalently bond with other side chains. Only with other cystines, makes a disulfide bond, stabilizes protein folding. |
| Importance of Serine, Threonine, and Tyrosine | Most likely to be phosphorylated because of the -OH groups in their side chains. They act as on/off switches. |
| Chromatography | Purify molecules from a mixture of cells. Separate by component of interest: size, charge, hydrophobicity, interactions with other molecules. Use multiple rounds. |
| Column chromatography | Uses beads and buffers to elute desired component. Ion exchange, Reverse phase, Gel filtration, Affinity. |
| Ion exchange chromatography | Positive beads attract negative beads, eluting positive proteins. Higher salt concentration prevents less negative beads from attaching, ensuring you get the MOST negative proteins on the beads. |
| Reverse phase chromatography | Hydrophobic beads attract other hydrophobic proteins, the salt stabilizes and there's less and less salt to elute more hydrophilic proteins at the bottom. |
| Gel filtration chromatography | Biggest go through fastest since small ones get stuck in a part of the gel molecule. By size. |
| Affinity chromatography | Beads have antigen, receptors, DNA protein, binding to the desired molecule. Fastest, but not always available. |
| How to design a chromatography experiment? | When affinity isn't an option, the rule is you want your molecule of interest to STICK to the beads, undesired stuff coming off. Strongest interactions stick. 1. Gel filtration for size 2. Reverse phase for hydrophobicity 3. Ion exchange for charge |
| What do proteins do? | Make things: synthesize DNA, RNA, other proteins. Destroy things: pathogens, old cell materials Cell communication Pretty much everything except the genetic material. |
| Why is protein structure important? | Structure defines ability to do its job and interact with other molecules. Most diseases happen from proteins without the right structure. Usually fold alone but chaperone proteins can help. |
| Primary structure | Amino acid sequence, peptide bonds between. |
| Secondary structure | alpha helix, b pleated sheets. R' groups on outside, hydrogen bonding between backbone. A helix: proximal bonds. B sheets: distant bonds. |
| Tertiary structure | Bonding between R' groups: hydrogen bonds, disulfide bonds between cystines, hydrophobic interactions, ionic bonds between acidic and basic side chains. |
| Quaternary structure | Multiple peptides joined together though hydrogen bonds or disulfide bonds. |
| 3 ways to model proteins | 1. Space filled 2. Stick form 3. Ribbon |
| Protein Domains | Regions with a specific function/location/shape. They direct movement, interact with other proteins, and activate proteins. Amino acids with -OH groups associated with phosphorylation (activation). Hydrophobic ones associated with hydrophobic molecules. |
| Transmembrane domains | Span the entire membrane, hydrophobic inside, philic outside. Have extra/intracellular domains. Intracellular activated by binding to a ligand and phosphorylating. |
| Channel proteins | Interact with water and other ions like K and Na. |
| Nucleosome | Histone octamer complex that compacts DNA. Outside is basic to attract negatively charged DNA. Domains are: 1 H1 2 H2B 2 H2A 2 H3 1 H4 |
| Helicase | Unwinds DNA. Part that interacts is positive to interact with negative DNA backbone. |
| Post translational modification FUNCTION | Activation/deactivation to change the peptides charge, polarity, or size. Happens to individual acid or multiple, changing the function of the entire protein. These are essential for proper protein folding. |
| Types of post translational modifications | Phosphorylation, acetylation, methylation, and glycosylation. |
| Phosphorylation | Add phosphate group to hydroxyl group on amino acid. Single charge changes the entire shape of protein. Kinase transfers group from ATP, making it ADP. Reverse: phosphatase uses hydrolysis to remove phosphate. |
| Acetylation | Histone acetyltransferase HAT transfers acetyl group HN=O-O from acetyl CoA to lysine, giving histone neutral charge, loosening DNA to be read. Histone deacetylase HDAC removes acetyl group. |
| Methylation | Methyltransferase transfers methyl group (CH3) to lysine, increasing the histones positive charge, packing DNA tighter preventing gene expression. |
| Glycosylation | Adding sugars with glycosylase to provide stability, solubility, and other functions. |
| Enzymes | Catalyze chemical reactions. End in -ase. Convert substrate into product. Produce building blocks and are involved in DNA replication (DNA polymerase), RNA transcription (RNA polymerase), and protein translation (ribosomes) |
| Models of enzymatic activity | Lock+Key Model: Perfect fit Induced Fit model: Enzyme and substrate negative and positive interactions change enzyme shape to fit substrate. |
| Detecting and quantifying enzymatic activity is done in 2 ways: | B-Galactosidase colorimetric assay and radioactive assay. |
| B-Galactosidase colorimetric assay | Normally substrate is lactose, produces galactose+glucose, but it can also use a synthetic substrate: ONPG to produce galactose+o-nitrophenol (yellow color). Use light absorbance, how bright is how much enzymatic activity. |
| Radioactive assay | Radioactive substrate (too many neutrons, makes it decay), measure product. Ingredients: Template DNA, radioactive nucleotides, sample of interest with the DNA polymerase will make radioactive nucleotides. |
| Steps of DNA Extraction | Break open cell Digest RNA w RNAse Separate DNA from protein w phenol extraction Precipitate DNA w salt+alcohol, centrifuge Dry DNA Dissolve DNA in buffer @8pH Ready 4 gels, cloning, etc. |
| Phenol extraction | Mix DNA:Phenol solution Centrifuge, DNA+RNA to aqueous phase Precipitate aqueous phase with ethanol |
| Why use alcohol in DNA extraction? | RNA+DNA soluble in water cuz polar. Alcohol less polar. Na+ in salt makes stable ionic bonds in DNA+RNA Forms DNA as insoluble salt |
| RNA Isolation | Freeze cells w liquid nitrogen+grind w pestle Lysis regent of guanidium+phenol denatures proteins Chloroform mixture separates phases w centrifuge Ethanol precipitate RNA |
| Gelelectrophoresis | Determines types of molecules, purity of molecules, and separates molecules. DNA moves (-) to (+) at rate depending on: charge, shape, and mass of fragment |
| Rules of gelelectophoresis | 1. Use mass ladder 2. Smallest DNA fragments go fastest 3. Smallest go to bottom 4. Brighter means more 5. Larger fragments brighter |
| Agarose gelelectrophoresis | Comes out in bands of different sizes+brightness Bigger DNA brighter+more dense cuz of ethidium bromide. |
| Ethidium bromide | Binds to and fluoresces DNA in UV light. Gets into rungs of DNA. |
| How many base pairs does agarose vs. polyacrylamide pick up on? | Agarose: 200-50,000 bp Polyacrylamide: 5-750 bp (picks up on single nucleotide differences) |
| Protein gel electrophoresis | Polyacrylamide (PAGE): separates on size, shape, and charge. No modifications to proteins. |
| SDS PAGE | Sodium dodecyl sulfate anionic chemical binds to proteins, giving all (-) charge. Separates on size. Beta-mercaptoethanol (BME) breaks disulfate bonds to denature protein's shape. Result: linear proteins, singular peptide chains. |
| Plasmids | Circular DNA found in bacteria used to store non essential genetic code. |
| Endonuclease | Cuts nucleic acid in the middle at specific sequences, internally. |
| Exonuclease | Cuts at the 5' phosphate group at the end of nucleic acids. Random sequences. |
| Restriction endonucleases | Defense mechanism in bacteria to cut up viral DNA injected by bacteriophage virus. |
| Restriction map | Shows position on plasmid where cuts are made to determine size of fragments. Use 1st digest enzyme, then 2nd. |
| Palindrome | DNA sequence that reads the same both directions: ACTG TCGA These are often restriction sites because nucleases make double stranded cuts. |
| T4 DNA Ligase | Creates covalent bond between 2 fragments to seal them. |
| Recombinant DNA | DNA combo from different organisms, human produced. Use to clone DNA. |
| Why clone DNA? | It produces a lot of DNA to manipulate and analyze. Allows for you to perform mutagenesis, express genes, and other manipulations. |
| Plasmid cloning vector components | BACTERIAL ORIGIN OF REPLICATION, SELECTABLE MARKER, enhancer element, minimal promoter, MULTIPLE CLONING SITE. |
| Bacterial origin of replication | Tells cell to plasmid to replicate |
| Selectable marker | BTA Lactamase giving resistance to Ampicillin. Ensures plasmid will keep the foreign DNA because that plasmid will be favored in ampicillin rich environment. Cells without the inserted DNA die. |
| Multiple cloning site | Lots of restriction sites (palindromes) to use different restriction enzymes so you can insert the DNA fragment you want. |
| DNA Blotting | Transferring nucleic acids/proteins from gel after electrophoresis to paper (a blot). |
| Probes for DNA blotting | Don't work well in gels since they can't get to target molecules. Paper easy for them to get to target. They look for specific nucleic acid sequences. |
| Hybridization | Annealing of the probe with the target strand. How? 1. Accurate base pairing 2. Antiparallel relative to each other. |
| How to make radioactive probes for hybridization | Purify target DNA seq. Use as template for probes. Nick target DNA w P23. Polymerase replicates DNA w radioactive phosphorus so you can see the seq of interest. |
| Steps to perform a southern blot | Electrophoresis DNA Salt solution blots DNA to nitrocellulose filter DNA transferred to filter, remove gel Hybridize w probes. Filter in ziplock Wash unbound probe. Remaining hybridized Expose filter to x-ray film, read audiogram |
| Steps of Northern blot | RNA isolation Agarose gel separate by size Nitrocellulose membrane blotting DNA probes nicked, bind in ziplock w UV/heat Wash unbound probes RNA visual on x-ray film adiogram |
| GAPDH | Loading control mRNA produced everywhere, shows us change from one sample to another, controls degradation of RNA. |
| Reading blot audiograms | Where more dense, more DNA, mRNA being expressed, protein being produced. Helps associate with diseases. |
| Western blotting | Detects specific proteins by using antibodies as probes. Secondary probes radioactively labeled. |
| In-situ hybridization | ISH detects nucleic acids in living/intact cells. Blotting requires lysing the cells, ISH does not. Learn about variety of cells in specific locations. Detects DNA/RNA, and uses fluorescent probes. |
| Sanger sequencing | Identify pathogens, fraternity, and genotype to determine disease susceptibility. Needs primers, fluorescent dye, and dideoxynucleotides |
| Dideoxynucleotide | DNA Nucleotide with 3 phosphate groups, except 3' -OH group is missing OTHER oxygen too, so only -H. "Chain terminator." Label base with colors, and can tell which base is which in seq through a gel. |
| Sanger sequencing unknown sequences | Insert fragments into plasmid vector, which seq is known so when it is cloned we can determine the base pairs of the inserted DNA from the organism. |
| Site directed mutagenasis | Changing 1 nucleotide in the primer so that it almost perfectly matches, but codes for a new protein, shows effect of mutations because 1 change in amino acid changes entire folding and function. |
| Quantitative PCR | Detects changes in copy number and how much DNA is present with taq-man probes that glow when a 2nd primer removes the dye from the primer, measure glowing number. |
| PCR uses | Polymerase Chain reaction. Detect specific DNA seq from mix, amplify DNA fragments, Modify DNA seq, sanger sequencing. |
| PCR ingredients | Thermostable DNA polymerase Primers Nucleotides Appropriate pH Divalent cations (Mg+) DNA template |
| Steps to PCR | 1. Heat at 95 C to separate/denature 2. Cool to 55-60 C depending on the primer so it can anneal to DNA 3. Primer extension at 72 C Repeat to amplify DNA |
| Taq polymerase | Found in bacteria that live in hot environments since ours can't work that hot. |
| Primers in PCR | ssDNA oligonucleotides complementary and antiparallel to target DNA, free 3' OH group to add nucleotides off of. Make A TON so they anneal instead of re-annealing the template. |
| Thermal Cycler | Cycles through the 3 temperatures around 40 times. |
| How to read PCR on gels | You should only get 1 band per ladder rung (sample). If multiple bands, it means the primers are annealing to other sequences besides the target. |
Created by:
lilelise
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