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*** Module 1*** ● PCR *** Know the steps of PCR*** one question asks about the first step Polymerase Chain Reaction= the process of copying DNA in the lab. With PCR you need: 1. Template DNA 2. Nucleotides (dNTPS) 3. DNA polymerase 4. DNA primers

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Steps of PCR 1. Denaturation (heated to 95C to separate it) 2. Annealing (reaction is cooled to 50C, primers stick to the DNA that you want to copy and ADD DNA polymerase 3. ELONGATION (reaction heated to 70C and DNA polymerase add nucleotides to building new DNA Strand “Have An Éclair” Heat Annealing Elongation ▪ For each round of PCR there are 2 created, continue to multiply by 2 1x2=2 2x2=4 4x2=8 8x2= 16 Ligase is NOT involved in PCR Remember the first step is denaturation / heating

● Base Excision Repair, know about it This is how you repair a mutation. Base excision repair is used to repair damage to bases caused by harmful molecules. You removed the base that is damaged and replace it. DNA Glycosylase see’s the damaged DNA and removes it. Then DNA polymerase puts the right base back in while DNA ligase seals it back up. Boom all fixed. ● Base excision repair (BER) removes a single nucleotide!!!!!! Only one base camp! ● Mismatch repair is the only one to occur during REPLICATION—DURING THE PROOFREADING. o During replication, DNA polymerase proofreads, but sometimes a mismatch occurs. So MMR removes a LARGE section of the nucleotides from the new DNA, DNA polymerase tries again. o Know what damage MMR repairs in DNA: G-C A-T ● NucleoTIDE excision repair- TIDE like the beach, sun exposure--- UV damage repair ⮚ A large section of nucleotides are removed, including the damaged portion, along with a few on each side. It’s then replaced by DNA polymerase. ● Homologous Recombination- repairs double stranded breaks—this is a last ditch effort ✔ Repair is made using a copy of the other strand of DNA and replacing it completely. ● Non Homologous Recombination- another double stranded break repair ✔ The cells put the ends back together before making sure they are correctly copied. This can lead to deletions/ insertions (Frameshift mutations). This is the last ditch effort and the body is willing to take that chance. ● Question about what DNA polymerase binds to DNA to make RNA ⮚ TRANSCRIPTION DNA takes the individual nucleotides and matches them to the PARENTAL sequences to ensure a correct pair. It must bind to RNA primer to work! ● DNA polymerase is needed for DNA replication ● 2-3 charts with questions about missense and nonsense mutations. Nonsense- stop all that nonsense, puts a stop codon in. Silent- Changes 1 nucleotide, but keeps the same amino acid

Missense- change in 1 nucleotide results in a completely different amino acid ● During RNA splicing, introns are cut out, the remaining Exons are joined together! o Know function of introns and extrons ● Know the process of DNA-RNA-mRNA, transcription, translation, etc. (3-4 questions about that!) Coding (non template)

NonComplementary

Template (non coding) Complementary

mRNA tRNA/amino acid

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Questions about changing from DNA template to mRNA Non template to complementary

● Central Dogma—Know it and know how to place it on picture

● Gene Expression: the ability to turn genes on or off ● Epigenetics: packing of DNA, where DNA is wrapped around the histones to make nucleosomes ✔ ↑ wide spread nucleosomes= the genes are ON ✔ ↓ tightly packed nucleosomes= the genes are OFF

*** Module 2*** ● Remembering amino acids o Polar amino acids- Polar bears like water! “OH, Look, it’s a Northern Hemisphere (NH) or Southern Hemisphere (SH) Polar Bear!!!” Polar amino acids = OH, NH, SH o Or use S.O.N o Non polar amino acids- hydrophobic- CH “Can’t Have” water o Ionized amino acids- the charged amino acids +/- attached to them. ● Know what ionized Alanine looks like It’s an amino acid, look for the R group, alanine is HYDROPHOBIC that has CH3 as it’s weak interaction. For ionized look for the one with the + or – charge.

● Misfolding of a protein structure in Alzheimer’s Disease o Caused by intracellular tangles and extracellular plaques (senile plaques) caused by ABNORMAL PROTEIN AGGREGATION. o TAU is fibrous material inside cells with this the connections are lost. This becomes defective and form filaments in the amyloid-beta is a large precursor protein in the cell. o Excess amyloid-beta is clearly linked to Alzheimer’s disease creating senile plaques. Starts in the hippocampus and moves up. ● Neurodegenerative protein aggregation: Alzheimer’s disease, the most common neurodegenerative disease. The formation of the aggregated amyloid-beta fibers is another characteristic of Alzheimer’s. The neurodegeneration and memory loss can be detected before the amyloid fibers accumulate in the brain. ● Chaperones- molecules that help denatured proteins in folding (they help newly formed proteins and can also help the misfolded ones) ● Know the levels of protein structure!!! (a question about the 3dimensional shape of a polypeptide, a question about the 2nd level—there are 3-4 questions on this topic) 1. Primary= chain of amino acids, peptide bonds forming a polypeptide chain. Covalent bond= strong and does not denature 2. Secondary= alpha helix and beta sheets, hydrogen bond, denatured by salt and pH change. Carboxyl grp and amino grp 3. Tertiary= 3-D shape (sickle cell, arthritis, hemophilia) Changes seen with ↑temp, salt, change in pH, and reducing agents 4. Quaternary subunit= more than one polypeptide, HgB Changes seen with ↑temp

● Primary formed by peptide bonds, secondary held together by hydrogen bonds. Hydrogen bonds formed from 2 polar amino acids!!!

● What structure would be unaffected by complete denaturation of multisubunit? Primary!!!! Peptide bonds are strong and covalent. ● Tertiary structure is HYDROPHOBIC, protein structure is stabilized primarily by the hydrophobic effect, disruption of the hydrophobic effect is the simplest way to denature—which is done by ↑temp from heating up. ● There is a question about methotrexate treatment in cancer, I think the question wanted to know how it affects enzyme activity. ------ methotrexate works by blocking an enzyme process in cancer cells so that they cannot grow ● Question about induced fit o The substrate and active site are somewhat complementary prior to the substrate binding, many enzymes will adjust their active site conformation slightly as the substrate binds to improve the fit. This is known as induced fit. When the molecule is recognized as the substrate, the enzyme will adjust to form itself around the substrate more tightly to facilitate a reaction ⮚catalyzes. Think about a friend giving you a hug. ● Know about substrate enzyme complex- like lock and key o Each substrate binds to an active site, this produces the enzyme-substrate complex. This geometrical design depends on noncovalent forces. ● Know about substrates, enzymes, products, and inhibitors: 2 questions w/ pictures you have to identify these o Substrate: molecule that an enzyme will bind preferentially to any other molecule. Each enzyme is specific for that substrate, it won’t react with molecules that are not its own substrate. o Active site: enzymes have an active site, which serves as the binding platform for its specific substrates and acts as the site of the chemical reaction. o Allosteric site: any site OTHER THAN the active site o Know what substrate | enzymes | active site look like on diagram ● Enzyme inhibition (competitive vs non-competitive) o Competitive- inhibitor- usually a molecule SIMILAR in structure to a substrate that can bind to an enzyme’s ACTIVE SITE even though the molecule is UNABLE TO REACT! o Non-competitive inhibitor- some non-competitive inhibitors attach to the enzyme at the allosteric site (a site other than the active site) ● Feedback inhibition (non-competitive) - know that once the end product builds up it will bind to the allosteric site on the first enzyme to stop (or inhibit) the pathway!! Meaning it binds to the enzyme to slow the making of the product. o Feedback inhibition is the result of the end product ● Question w/ pics of amino acids where you pick out the non-polar (CH) amino acid Look for CH, CH2, CH3 🡪 these are weak interactions that tend to go in the middle of a protein (aggregation) and are affected by temperature change. ● What part of a phospholipid is hydrophobic? The tails ● Peptide bond- peptide bonds form between 2 amino acids via DEHYDRATION reaction. During the reaction a water molecule (H20) forms from the oxygen of a carboxyl grp and 2 hydrogens from an amino grp. As water forms, the carbon atom of the carboxyl grp and the nitrogen atom of the amino grp become bonded together. This bond between the two amino acids is called a peptide bond.

● Activation energy: There is a question where you must label the area of activation energy ▪ The energy required to start a reaction ▪ Enzymes help to catalyze the reaction—they speed it up!! ▪ Enzyme action helps reactions happen quickly by lowering the activation energy

*** Module 3 *** ● Question about cooperative binding of hemoglobin: o the 4 subunits of hgb work together. When 1 molecule of O2 leaves, the other 3 tend to drop off too. When 1 O2 is picked up, the others tend to pick up too. This is how it’s picked up in the lungs and dropped off in the tissue. o Cooperative binding: O2 will bind quicker when there is another O2 molecule already there. ● Carbon Monoxide (CO) poisoning ▪ CO binds to the same grp as O2 but it does so more strongly (200x stronger). If there is CO around, O2 is picked up and is not dropped off. This is why CO poisoning is so harmful. The patient is unusually pink in color. ▪ CO stabilizes the R-state by CO binding to the same Fe in HgB and changes the heme shape. ● Know about 2,3BPG and O2 affinity for fetus ⮚ Fetus has higher affinity for O2 than mom does ⮚ Affinity is the stickiness for O2 ⮚ A human fetus needs a higher affinity for O2 to get it from its mother ⮚ The mother’s 2,3BPG is higher so their affinity for 02 is lower⮚ the mom becomes deoxygenated ⮚ 2,3BPG stabilizes the T-state (increased in pregnant women) ● Question about how hgb structure is affected in absence of O2 o This is found in the muscles—the T state. The tense state, the heme is bent – FREEZE! Hold your hands up! Ph is lower ● Know about hemoglobin and myoglobin ▪ Hgb- has low affinity for O2 and transports it throughout the body ▪ Myoglobin- has high affinity for O2 and stores it. Not affected by changes in ph 7 ● How pH influences o2 saturation of hgb at the same partial pressure—BOHR Effect ⮚ Left shift- ↑affinity for o2 ⮚ Right shift- ↓affinity for o2 Bohr Effect (The way that I remember this is thinking of a exact opposite.)

blinker signal, Bohr effect is the

● What makes LDL? High cholesterol ✔ Made of a 4 ring structure. AKA sterols (cholesterol, cortisol, and testosterone.) ✔ It expands the range of the membrane’s fluid; greatest membrane fluidity ✔ Solidifies at ↑temp ✔ Cholesterol is needed to synthesize Vit. D in the skin, cholic acid (a component of bile), and steroid hormones ✔ The steroid hormones include testosterone and estrogen- which promote growth and development of sex characteristics. Also, cortisol which is released in response to stress and promotes glucose synthesis in the liver

● What can show a MI??? Myoglobin Myoglobin is a protein w/ a single subunit containing a heme and iron. While myoglobin is normally contained only in muscles, it can be released into the bloodstream if there is Muscle damage (MI). For this reason, measuring the levels of myoglobin in the blood can provide evidence of rhabdomyolysis, a mild heart attack, or a muscular degenerative disease.

*** Module 4 *** ● Know about the anabolic pathway (2-3 questions on this) ▪ This is where there is no O2, the ETC cannot work. When NAD+ runs out it, it’s then made with fermentation. This is where 2 pyruvate are broken down into lactate and this generates the NADH to NAD+ that was used during glycolysis to make small amt of 2 ATP ▪ The Cori Cycle uses the lactate made by fermentation in the liver and uses gluconeogenesis to convert two lactate to glucose ⮚ 2 ATP are made with each cycle but it uses 6 ATP to do this. Net loss of -4 ATP ● Know anabolic and catabolic pathways: Anabolic- ends in GENESIS ⮚ Builds up – requires energy to synthesize smaller into larger molecules Catabolic- ends in LYSIS ⮚ Breaks it down – those that GENERATE energy by breaking down larger molecules. Both of these are required to keep the cell’s energy in balance_____________ ● How does the Cori Cycle supply for the RBCs? ✔ RBCs don’t have mitochondria ✔ The Cori Cycle takes up lactate from the blood and uses gluconeogenesis to convert 2 molecules of lactate back into glucose. ✔ The byproduct of anaerobic glycolysis, lactate, diffuses into the blood and is taken up by the liver, where it is converted back to pyruvate by the enzyme lactate hydrogenase—the newly formed glucose is released into the blood to be used once again for energy by the RBCs ✔ Cori Cycle supplies glucose to the RBCs ● The Cori Cycle (recycle) When there is no O2, pyruvate from glycolysis is converted to lactate and put into the blood, the LIVER picks up the lactate and uses gluconeogenesis to convert 2 molecules of lactate into 1 glucose ● Which molecules starts the Citric Acid Cycle? Acetyl CoA

● CAC – NADH and FADH2 are needed to continue with aerobic metabolism! ● Effect of glucagon on carb metabolism⮚ glucose conversion into glycogen for later use during hypoglycemia. Glycogenesis: the formation of glycogen from sugar ● Questions about lactic acid fermentation▪ If O2 is scarce (anaerobic conditions) for exp. In skeletal muscle fibers during strenuous exercise- then pyruvate is reduced via an anaerobic pathway by the addition of 2 hydrogen atoms to form lactic acid (lactate). This is known as fermentation. ▪ The formation of lactate regenerates the NAD+ that was used during glycolysis, thus allowing glycolysis to continue while making small amt of ATP for the cell. ● Substrate level phosphorylation is the only way to make ATP w/o O2 present. ● GluT Transoporters▪ Act as a channel to move glucose into the cell through facilitated diffusion and active transport ▪ Insulin is what moves the GluT Transporters to the cell membrane so this can happen ● Metformin decreased amp in the liver muscles cells. Metformin exerts its antihyperglycemic effect mainly by inhibiting liver glucose output. Both glucogenolysis and gluconeogenesis are reduced by metformin. ● Questions about a runner!!!!!!! o Eating high carbs the day BEFORE a race (carbs w/o exercise) Gluconeogenesis – build up for race day o Eating high carbs the day OF a race is glycolysis because it’s burned right away o A person running and losing needs to tap into glycogen stores and stimulate glycolysis ● G Words ▪ Glucagon- activates when BS is low--Glucagon stimulates glycogenolysis to release glucose for the liver to export to the bloodstream when blood sugar is low. ▪ Gluconeogenesis- makes glucose from non carb (pyruvate, lactate, glycerol) NADH is oxidized into NAD+ for glycolysis (Cori Cycle) ▪ Gluconeogenesis- gluco (glucose) neo (new) genesis (make) – anabolic pathway w/ a peptide bond ▪ Glucogenesis- make glycogen ▪ Glycolysis- triggered by carb intake during exercise ▪ Glycation- caused by increased BS can lead to AGE (Advanced Glycogen End products) which cause proteins to shrink. ▪ Glucose- 6 carbon sugar: fuel our tissues most prefer ▪ Glycogen- storage form of glucose ▪ Glycogenolysis- breakdown of glycogen to release glucose (fasting)

● By contrast, glucagon is the hormone that signals the hungry state. Thus, it switches cells (especially liver cells) to a program of releasing stored energy.

*** Module 5 *** ● Beta oxidation happens in the mitochondria matrix ● Molecules of Beta oxidation produced are: 1. ATP This happens after 1 round of 2. NADH Beta Oxidation 3. FADH2 4. Acetyl Co A ● To calculate: 1. Count the # of Carbons divide / by 2 2. Subtract 1 from this ● Beta oxidation is fatty acid breakdown ● Beta-oxidation is stimulated by glucagon. Acetyl-CoA is produced by beta-oxidation. ● Pay attention to what the question is asking in terms of # of Acetyl CoA and # of rounds. It may be turned around. ● Roughly a 16 carbon fatty acid yields 100 ATP vs 1 glucose yields 30 ATP ● MCADD- enzyme used to oxidize medium chain (carbons 4-12) is defective. ** question on this ** ⮚ Diet needs to be slow released carbs to maintain energy levels ⮚ Carbs over 12 can be fatal ⮚ Must avoid fasting ⮚ Avoid fats and eat complex carbs (slow releasing) ● Be able to locate an Omega 3 and Omega 6 fatty acid

These are the alpha and Beta and are read from R to L

This is the Omega (it’s read from L to R) ● If the first double bond occurs btw the 3rd and 4th carbons, it’s an omega 3 fatty acid ● If the first double bond occurs btw the 6th and 7th carbons, it’s an omega 6 fatty acid ● Unsaturated bonds most likely to be liquid at room temp. ⮚ There is a question on this ▪ Don’t stack well ▪ Lower melting point ▪ Ex: olive oil ● Where are fatty acids catabolized? The fatty acetyl CoA molecules enter the mitochondrial matrix and begin the cycle of beta oxidation!!! ● Fatty Acid Synthesis __ BUILDING UP This happens in the cytosol ▪ This how to build up fatty acids ▪ You build up two carbons from one acetyl CoA ▪ Takes place in the cytoplasm ▪ Produces ATP ▪ Acetyl-CoA is converted to citric acid for export out of the mitochondrion then reconverted to acetyl-CoA in the cytoplasm ● There are pictures of Amino Acids and you will have to pick the hydrophobic with CH— remember Can’t Have= CH ● Trans- hydrogens on opposite sides of the double bond making the carbon chain straighter. o An unsaturated fatty acid of a type occurring in margarine and manufactured

cooking oils as a result of the hydrogenation process, having a trans arrangement of the carbon atoms adjacent to its double bonds

● Know TRANS fatty acid vs CIS configuration!!!!! ⮚ CIS configuration- both hydrogens on the same side. - Takes less heat to melt because it’s not packed tightly - The asymmetry forces a kink or bend in the carbon chain, making it difficult for the fatty acids in the CIS configuration ⮚ Trans configuration- the hydrogen is OPPOSITE sides of the double bond!! Trans fatty acids pack TIGHTLY like saturated fatty acids and so have a higher melting point than the same fatty acid in the CIS configuration. ● A person wanting to limit fat question. ⮚ the Triglycerides picture This is the greatest reserve of energy – it has a glycerol backbone with 3 fatty acid chains

● Eicosanoids- arachidonic acid- 20 carbon fatty acid (polyunsaturated- Omega 6 fatty acid) ⮚ Aids with inflammation, fever, immunity, and blood coagulation

● Know the fluid mosaic model, the contents and the parts of it: ⮚ Fatty acids and triglycerides have a leaky membrane (because it has spaces) and phospholipids are backed tightly with an impermeable membrane ⮚ Remember, Phospholipids have 2 fatty acid tails

The polar heads are hydrophilic (they like water)

The tails are hydrophobic (they are afraid of water)

There is a question on this topic where you have to recall what components are in the Fluid mosaic, I think it was asking which of the answers DID NOT BELONG. Question about why amylose is more easily digestible than cellulose: The difference between amylose and cellulose is the way in which the glucose units are linked; amylose has αlinkages whereas cellulose contains, β-linkages. Because of this difference, amylose is soluble in water and cellulose is not. ❖ Use your white board!!! When you can dump it on the board, erase it, and dump it again it really helps you. ❖ Some ideas to be able to dump: o The picture of complementary/ non complementary DNA/RNA o Process of glycolysis (talk to Janet at the helpdesk, she is great!!!!) o T-state and R-State characteristics (these can be found in other dump files, but Janet at the helpdesk is the b...