Biochemistry Study Guide: Understanding Biological Molecules & Metabolism, Summaries of Biochemistry

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J. Berg, G. Gatto, J. Hines, J. L. Tymoczko,

L. Stryer,

Biochemistry: study guide

BIOCHEM – EXAM 1

Biochemistry – study of life processes CHAPTER 1 - MICHEL

• 2 types of biological molecules
  1. macromolecules proteins, RNA, DNA
  2. metabolites = chemically transformed during biological processes glucose, glycerol Key metabolic transformations are common amongst a wide range of organisms which leads us to believe that all organisms share a common ancestor and have then evolved to today’s diverse life forms
• STRUCTURE DETERMINES FUNCTION DNA is prime example
• DNA *stores genetic info *linear molecule *backbone made up of repeating sugar-phosphate units *4 bases A denine and Guanine = purines, pair with T hymine and Cytosine = pyrimidinesvia H bonds (double and triple respectively) *can base pair in a test tube 1:1 ratio of strands, 25 degrees C, 1M NaCl
• All biological molecules follow basic chemical principles *Bonding - covalent - non-covalent electrostatic forces, H-bonds, Van der Waals, hydrophobic interactions *Thermodynamics *Acid-Base Reactions *pH and buffers
• Covalent bonds = strongest bonds electrons shared b/w 2 atoms *resonance structures = more than 1 pattern of covalent bonds seen, stronger bond than single but weaker than double
• Non-covalent = positive and negative charge NaCl *governed by Coulomb’s law E = kq 1 q 2 /Dr^2 – D = dielectric constant (effect of solvent) *H-bonds = electrostatic interaction where H shared b/w 2 electronegative atoms (N or O) - donor = contains atom which H is more tightly linked and H atom itself - acceptor = atom that’s less tightly linked to H atom *Van der Waals = distribution of electrons around at atom fluctuates such that transient asymmetry forms allowing 2 atoms to come closer together than would be predicted distance = Van der Waals distance - NRG is very low *Hydrophobic effect = electric dipole forms where 1 atom draws e-s from other atom which leads to “-“ charge around that atom and “+” charge around atom(s) losing electrons ex: H 2 O

solution will drop much more rapidly buffers function best close to pKa of acid component *physiological pH = 7.4 phosphate solutions are good buffers for physiological systems If you decrease the pH by 1 unit, the ratio of ion to acid decreases ten-fold b/c it is proportional to log = power of 10 decrease by 6 units = ratio changes 10^6 fold

• Dogma of molecular biology = DNA RNA Protein (2nd^ dogma = amino acid sequence results in only 1 type of protein) *particular amino acid sequence dictates protein fold *3 bases along a strand of DNA (codon) determine which amino acid is formed *relationship b/w DNA and amino acid = the GENETIC CODE CHAPTER 2 – BUTKO
• primary structure = amino acid sequence, secondary = formed by a.a. interactions, tertiary = interactions b/w secondary structures, quarternary structure = only occurs in proteins w/ multiple subunits
• Proteins = most versatile macromolecules in living systems *functions: - catalysis - transport and/or storage - immune protection - mechanical support - movement generation - transmission of nerve impulses - control (of growth, differentiation, or of other protein’s function) *properties: 1) linear polymers of amino acids 2) fold into 3 - D structures 3) contain diff. functional groups – thiols, alcohols, carboxylic acids “+” charged, “-“ charged, or neutral at pH 7.
  4. often interact w/ other macromolecules – proteins, DNA, RNA, etc translates into specific function
  5. some are ridid, some are flexible
• Alpha C = central C that carboxylic acid, H, and R group are attached to *amino acids are chiral = 2 isomers L and D all proteins are made up of L isomers *L isomers = if go CCW on alpha C, you go from highest priority to lowest (NH3 COO- R H)
• Amino acids *at diff pHs the charge on a.a. changes at neutral pH (7.4) exist as zwitterions = amino is “+” and carboxylic is “-“, at low pH both groups are protonated = net “+” charge and at high pH both are deprotonated = net “-“ charge *pKa carboxylic = 2 and pKa of amino = 9

*are grouped together based on physical-chemical properties

• Nonpolar/hydrophobic (aliphatic) side chains = cluster together in water glycine (G), alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M) = contains S + non-polar side chain, Proline (P) = has aliphatic side chain bonded to N alpha C this structure determines its function = used where a turn is necessary in proteins
• Aromatic side chains = Phenylalanine (F) = hydrophobic, Tyrosine (Y) and Tryptophan (W) = less hydrophobic b/c of OH and NH groups *cause absorption
• Polar, uncharged side chains = Serine (S), Threonine (T), Asparagine (N), Glutamine (Q), Cysteine (C) *OH groups make S and T very hydrophilic as well as gives them a dipole moment *Carboxamide at end of R group on N and Q hydrophilic, N and Q uncharged derivatives of aspartate and glutamate (they have carboxylic acid at end of R group instead of carboxamide, also O and N bonds can H-bond *SH group at end of C = sulfhydryl or thiol group very reactive by forming disulfide bonds or binding metal ions
• Polar, charged - basic side chains = “+” charged at neutral pH, highly hydrophilic *Lysine (K) = primary amino group, positively charged at neutral pH *Arginine (R) = guanidinium group, positively charged at neutral pH *Histidine (H) = imidazole group, pKa =6 so can be “+” charged or uncharged at neutral pH depending on environment
• typically found at active sites of enzymes
• only a.a. that can change charges at neutral pH releases protons Histidine ionization how histidine binds ordepending on pH
• Polar, charged – acidic chains = highly hydrophilic, “-“ charged at neutral pH because give up proton at low pHs *Aspartate (D) and Glutamate (E)
• Some amino acids are unsuitable for proteins b/c of undesirable cyclization Homoserine has extra CH2 group that allows it to form a 5 - membered ring which can cleave a peptide bond

*antiparallel = straight H bonds, parallel = angled H bonds which makes parallel sheet less common *can also have mixed beta-sheet *can be straight or twisted (not in same plane) *fatty acids are rich in Beta sheets *Reverse turns = way in which peptide changes direction H bond forms b/w CO group of residue i of polypeptide chain and NH group of residue i + 3 to stabilize turn *Loop = another way to make turn in polypeptide much longer and more flexible than reverse turns *Fibrous proteins = structural support for cells and tissues, ex: alpha-keratin = found in wool and hair

• coiled-coil protein = 2 right handed alpha-helices that entwine to form stable structure
• very extended structure
• central region of a.a. w/ a sequence of 7 a.a. heptad repeat = every 7 th^ amino acid is Leu and 2 alpha-helices are kept together by van der Waals interactions and hydrophobic interactions reason you can stretch wool is b/c you can disrupt the van der Waals interactions, wool also has disulfide bonds that keep it together
• Collagen = most abundant protein in mammals, makes up skin, bone, tendon, cartilage *rod-shaped, very long and skinny, 3 helical polypeptides ~1000 residues long *every 3 rd^ a.a. = glycine b/c no space in center of helix *fold not determined by H bonds, but in how Pro rings sterically repulse each other forms superhelical cable stabilized by H bonds b/w each strand, φ and ψ dictate a.a. arrangement
• Tertiary Structure = water soluble proteins fold into compact structures w/ non-polar cores *X-ray crystallography and NMR = techniques used to obtain 3 - D structures *in an aqueous environment, hydrophobic a.a. must be excluded from water, which dictates protein folding most alpha helices and beta sheets = amphiphillic one side hydrophobic and other hydrophilic *porins = exception to hydrophobic interior and hydrophilic interior b/c have hydrophilic channel *certain types of structures or motifs are found in proteins w/ similar functions DNA binding have helix-turn-helix motif *domain = repeating set of structured polypeptide units
• Quaternary Structure = polypeptide chains can assemble into multisubunit structures *multiple polypeptide chains and how they relate to each other *ex: Hemoglobin = oxygen carrying protein in blood – is a tetramer w/ 2 alpha subunits and 2 beta subunits
• AA Sequence Determines 3 - D structure *Christian Anfinsen worked with unfolding and re-folding Ribonuclease (124 aa w/ 4 disulfide bonds)
• added Beta-mercaptoethanol (cleaves disulfides reversibly) and 8M urea (unfolds protein so that it is in random

coil configuration lost enzymatic activity

• dialysis into buffer w/o urea or Beta-mer… resulted in protein re-folding
• conclusion: information required for protein catalysis comes from amino acid sequence + sequence determines fold!! *if denatured ribonuclease is re-oxidized while still in urea and then refolded by dialyzing urea away, result is scrambled ribonuclease structure that shows limited enzyme activity wrong cysteines are oxidizedand paired *if trace amounts of Beta-mercaptoethanol are added, ribonuclease adopts correct, active structure every protein has unique tertiary structure, and if they misfold they can be linked to diseases like Parkinson’s and Alzheimer’s *diff aa’s have diff affinities for diff 2ndary structures Ala, glu, leu = alpha-helix; val, ile = Beta-sheet; Gly, pro = turns *same sequence can form diff 2ndary structures ie alpha-helix in 1 protein and beta-sheet in another *Protein mis-folding and disease
• PrP = protein normally found in brain, mostly alpha-helices infected PrP proteins have Beta-strands from one protein link w/ Beta-strands of another to form Beta-sheets = amyloid fibrils interactw/ normal proteins and lead to neurological diseases *fibrils are not soluble and lead to cell death in cells that harbor them *Protein folding = all or none situation w/ cooperative transition conditions that disrupt any part of the protein means entire protein will unravel
• folding doesn’t happen RANDOMLY Levinthal’s Paradox = it would take millions of years if proteins folded completely randomly but in reality, they fold in milliseconds or seconds parts of protein fold correctly and then when another part isn’t folded correctly,the protein “remembers” that correct portion when it re-arranges to find correct conformation of whole protein *Protein Modifications:
• covalent modifications can occur after protein is synthesized adding OH, COO, POO or other groups helps to stabilize protein further
• SUMMARY: Primary Structure = amino acid sequence Secondary Structure = spatial arrangement of amino acids that are nearby in sequence Tertiary Structure = spatial arrangement of amino acids that are far apart – overall structure w/ all atomic coordinates Quaternary Structure = spatial arrangement of the individual subunits (from tertiary structure) and the nature of their interactions, simplest example = dimer

separate things that are very similar, couldn’t do w/ previous methods

• Determining Protein Purity
  1. Gel Electrophoresis = uses polyacrylamide gel which serves as a molecular sieve that enhances separation small molecules move much more swiftly thru the gel *process: electric field applied that forces the molecules to migrate down the gel, smaller molecules move more rapidly as they have a lower “f” (frictional coeffictient v = Ez/f) *proteins are then stained after electorphoresis w/ coomassie blue or silver (0. micrograms and 0.02 micrograms)
  2. Isoelectric Focusing = separate by electric charge contents of acidic and basic residues *pI = pH at which protein’s net charge = 0 *gel has pH gradient and migrate to their pI and then form bands in the gel good to verify gel electrophoresis
  3. 2 - Dimensional PAGE = combine isoelectric focusing w/ SDS-PAGE separates proteins in horizontal direction according to isoelectric point and vertical direction on basis of mass
• Purity then Quantitatively Evaluated = measuring total protein, total activity, specific activity (important!!), yield, and purification level *specific activity increases as protein is purified
• Assay to Determine that Correct Protein has been Isolated or Understand Protein Function *Ultracentrifugation and mass determination = use sedimentation coefficient to quantify rate of movement of particle thru a medium using mass, partial specific volume, density of the medium, and the frictional coefficient smaller S value = slower a molecule moves thru a medium - large parts of protein sediment quicker than the smaller/lighter parts - proteins come in all diff shapes and sizes and densities *Zonal Centrifugation = used to separate proteins w/ diff sedimentation coefficients *Determining amino acid sequence - heat in 6M HCL to hydrolyze = breaks peptide bonds - use ion exchange chromatography to separate and identify revealed by elution volume = volume of buffer used to remove amino acid from column
  • add ninhydrin (or fluorescamine = detects aa at lower levels) to quantify now have which aa and what concentrations they are at after these steps need to find sequence
  • use Edman Degradation to determine sequence = removes one residue at a time from the amino end of peptide *PTH comes in and binds with N-terminal aa, acidic conditions the PTH releases w/ N- terminal aa from chain, then process is repeated until you have no more aa left on peptide identify each aa with HPLC
  • doesn’t work that well for larger proteins!!!

used

• larger aas are proteolyzed into smaller sequences and then Edman Degradation is *are certain proteolytic enzymes that cleave peptides at very specific residues at specific points if given chart be able to use!! *polypeptide chains linked by disulfide bonds need to be separated by reduction w/ thiols and to prevent residues from recombining they are then alkylated so they can then be sequenced with enzymes *Once we know sequence:
• we can relate our sequence to similar proteins (homologs) maybe can tell us something about protein’s functions
• can compare to proteins of diff species evolutionary information
• can use this info in search for novel drugs ROLE OF IMMUNOLOGY IN PROTEIN DETECTION
• an antibody (or immunoglobin (IG)) = protein that is synthesized by animal in response to foreign substance (antigen)
• we can make antibodies to peptides or proteins where protein or peptide = antigen
• Antibody *made up of 4 chains – 2 heavy and 2 lighter – that are connected by sulfide bonds *2 types:

1. polyclonal = can bind to multiple sites on an antigen
2. monoclonal = only recognize 1 specific site on antigen *antibodies can be used as specific analytic reagents to quantify the amount of a protein or other antigen Enzyme-Linked Immunosorbent Assay (ELISA), 2 methods: 1)Indirect = antigen coated well specific antibody introduced bind to antigen enzyme-linked antibodies to human antibodies are introduced and bind to the specific antibody substrate is then added and converted by enzyme into colored product w/ rate of color formation proportional to amount of specific antibody
3. Sandwich = monoclonal antibodies in well specific antigen added 2 nd^ monoclonal antibody added, linked to enzyme, binds to immobilized antigen substrate added and converted by enzyme to colored product

• medicinal application = HIV test indirect ELISA = viral core proteins that are part of HIV are put on bottom of well, antibodies from person being tested are added, if they have HIV they will have antibodies that recognize viral core proteins, enzyme linked antibodies are added followed by substrate which is then detected *Applications of Antibodies 2 Western blotting = very useful if you have a small quantity of protein or need to resolve a single protein from a mix of them

• electrons scatter x-rays = amplitude of wave scattered by an atom is proportional to its number of electrons, ie a C atom scatters 6x as strongly as an H atom does
• the scattered waves recombine = each atom contributes to each scattered beam, scattered waves reinforce one another at the film or detector if they are in phase there and they cancel one another out if they are out of phase
• the way in which the scattered waves recombine depends only on the atomic arrangement *different degrees of resolution can be achieved based on amount of data used in the Fourier synthesis
• NMR = reveals the structures of proteins in solution *solution must be very dense in order for NMR to work *based on fact that certain isotopes’ nuclei are intrinsically magnetic spinning of a proton generates a magnetic moment, which can then take 1 of 2 orientations alpha or beta when outside magnetic field is applied *alpha has lower NRG, but can be raised to beta state and this transition b/w spin states produces the NMR resonance line on the spectrum *2-D spectrum displays pairs of protons that are in close proximity point is to detect the location of atoms relative to one another in 3 - D structure of the protein CHAPTER 12 – BUTKO
• Membranes = act as a barrier and a bridge b/w cell and environment (selective permeability) *function in NRG conversion and storage as well as information transduction *features:
• sheet-like structures 2 molecules thick that spontaneously form closed boundaries
• non-covalent assemblies of lipid and protein + covalently linked carbohydrate moieties
• asymmetric = diff lipids in inner and outer layer
• fluid in 2 dimensions
• most often electrically polarized – membrane potential
• specific proteins mediate distinctive functions
• mimic bubbles flexible, weak
• Fatty Acids = key constituents of lipids *16:0 # C:# unsaturated C (double bonds) *vary in chain length and degree of unsaturation however, most are fully saturated
• Lipids = chemically diverse small biomolecules that are insoluble in water but highly soluble in organic solvents *biological roles = fuel, NRG stores, signal and messenger molecules, components of membranes *types = fatty acids (toxic if floating inside of cell), triacylglycerols (completely non-polar), waxes (long chains of C),

membrane lipids phospholipids, glycolipids, sterols *Phospholipids = glycerol backbone attached to fatty acids on left phosphate on right which is connected to an alcohol

• exception = sphingomyelin sphingosine (amino alcohol w/ long, unsaturatedhydrocarbon chain) is the backbone, not glycerol; sphingosine is also only attached to 1 fatty acid *Glycolipids = don’t contain a phosphate sugar moiety always faces outside of the cell
• backbone = sphingosine are like sphingolipids w/o phosphate
• backbone w/ 1 fatty acid on left and connected to sugar unit on right (extracellular side) *Sterols = backbone is 4 linked hydrocarbon rings linked to steroid at one end and other end has hydroxyl group (polar)
• in membranes, molecule is orientated such that it is parallel to fatty acid chains of phospholipids and hydroxyl group interacts w/ nearby phospholipid head groups
• Phospholipids and Glycolipids = form bimolecular sheets in aqueous media *membrane lipid = amphipathic molecule containing a hydrophilic (head) and hydrophobic (tail) moiety membranes form based on the principle of hydrophobic interactions
• van der Waals, electrostatic forces, and H-bonding forces play 2ndary role to forming supramolecular lipid structures
• micelles, bilayers, and liposomes are formed molecular shape of lipid determines which form it will be phospholipids form bilayers
• liposome = small aqueous compartment surrounded by a lipid bilayer
• increasing temp or adding unsaturated fatty acids increases fluidity
• lipid bilayers = are very extensive, close on themselves, and are self-sealing
• Lipid Bilayers = highly impermeable to ions and most polar molecules selective permeability *permeability of small molecules = correlated w/ their solubility in non-polar solvent relative to their solubility in H2O *proteins carry out most of the membrane processes = need to interact w/ lipid bilayer
• ex: integral (can’t dissociate from membrane so interact extensively w/ hydrocarbon region via nonpolar interactions), peripheral (can dissociate from membrane – most bind to surfaces of integral proteins, but some interact w/ polar head groups of lipids via electrostatic interactions and H-bonding interactions that can be disrupted by change in pH or salts) *proteins can span the membrane w/ alpha helices or Beta strands bacteriorhodopsin = made up almost entirely of alpha helices, uses light NRG to transport protons from inside cell to outside which generates proton gradient used to form ATP membrane spanning proteins = made up almost entirely of alpha helices
• Beta strands typically make up channel proteins like porins (transfer H2O inside and outside cell) arrangement of

*pumps = require NRG usually in form of ATP to transport molecules = active transport *channels = thermodynamically “downhill” flow no NRG needed = passive transport and facilitated diffusion

• move from high electrochemical potential to low
• Membrane transport is active or passive *simple diffusion = down the concentration gradient directly thru the lipid membrane only lipophilic (nonpolar) molecules *facilitated diffusion/passive transport = move down the electrochemical gradient, but thru a channel b/c cannot pass thru hydrophobic membrane ions and small polar molecules *active transport = move against the concentration/electrochemical gradient (low to high) with an input of NRG
• NRG stored in concentration gradients can be quantified *for a charged species, unequal distribution across the membrane generates an electric potential *for uncharged species is no electric potential generated so ΔG = RT ln (c2/c1) *for charged species need to take into account membrane potential ΔG = RT ln (c2/c1)

• ZFΔV *transport process must be active when ΔG = positive, and can be passive when ΔG = negative

• 2 Families of Membrane Proteins Use ATP hydrolysis to pump ions and molecules across membranes *P-type ATPase = Ca2+, Na+K form phosphorylated intermediates and couple that w/ conformational changes to pump ions across membranes
• 2 Ca2+^ ions bound to ATPase ATP binds ATP cleaved and P transferred to diff site, ADP released causing conformational change change causes Ca to be released on opp side of membrane Pis hydrolyzed and enzyme reverts back to original conformation *certain steroids digitalis inhibit ATPase by blocking dephosphorylation *ABC (ATP-binding cassette) transporters = members of P-loop NTPase superfamily – multidrug resistance protein
• have 2 transmembrane domains and 2 ATP binding domains (ATP binding cassette)
• process: begin closed free of ATP and substrate, can interconvert between closed and open forms substrate enters central cavity inducing conformational change which increases affinity for ATP ATPbinds to changing conformations so 2 domains interact change causes substrate to be released outside of cell ATP hydrolyzed and ADP released and transporter resets for another cycle
• Lactose Permease = an archetype of 2ndary transporters that use 1 concentration gradient to power the formation of another

*are antiporters, symporters, and uniporters *antiporters = downhill flow of 1 species to move another species uphill in opp direction across membrane *symporters = use flow of 1 species to drive flow of diff species across membrane in same direction *uniporter = like ion channels, transport specific species in either direction governed by concentrations of that species on either side of the membrane *ex: symport = lactose permease 2 halves oriented open to outside cell binding pocket protonated so binds lactose structure everts so facing inside cell lactose released into cell proton released complex everts back to facing outside to complete cycle

• Specific Channels can Rapidly Transport Ions Across Membranes *Action Potentials = electrical signal produced by flow of ions across plasma membrane - interior = high [K+] and low [Na+] - once membrane potential depolarized beyond threshold value (- 60 mV) potential becomes positive very quickly and then quickly turns back negative
  • depolarization leads to increased permeability to Na+ ions so they flow in very quickly further depolarization eventually membrane potential rapidly changes to positive more permeable to K+ ions membrane potential returns to negative value *Patch-Clamp Modes = proved ion channels specific for Na and K existed
    • patch pipette attaches to cell membrane and thru suction forms seal seal ensures an electric current flowing thru pipette membrane broken by increased suction low-resistance pathway b/w pipetteand interior of the cell activity of cell monitored while a known voltage is applied across membrane (flow of ions thru a single channel and transisitions b/w open and closed states of channel monitored) *Potassium Channel
    • parts of the AA sequence are the same in prokaryotes and eukaryotes = line the pore opening of the channel
  • however, calcium and sodium channels have a sequence that prokaryotes do not contain and is thought to control the opening and closing of the pore (S1-S6 segments = membrane spanning alpha-helices)
  • Selectivity filter = what makes it so K+ comes into the channel opposed to Na+ and other “+” charged ions *K+ is surrounded by H2O when it comes in to pore and NRG needed to dehydrate it is less than NRG gained when K+ H2O bonds are replaced by carbonyl group interactions to bind ion to channel *overall: NRG gained when dehydrated and bound to channel

returns to original closed state

• potassium channel = starts closed like Na+ and stays closed while Na+ open, then opens when Na+ inactivated, stays open until repolarization, and then is inactivated
• Gap Junctions or cell-to-cell channels = serve as passageways b/w interiors of contiguous cells *small hydrophilic molecules and ions can pass thru these junctions *all polar molecules less than 1 kd can readily pass thru cell-to-cell channels inorganic ions and most metabolites (sugars, AA, nucleotides) can flow b/w interiors of cells joined by gap junctions, while proteins, nucleic acids, and polysaccharides are too large *very important for intercellular communication *diff from other membrane channels:
• transverse 2 membranes, not 1
• connect cytoplasm to cytoplasm – unlike others that connect to extracellular space
• are synthesized by 2 diff cells *once formed stay open for sec or min *controlled by membrane potential and hormone-induced phosphorylation *closed by high [Ca2+] and [H+]
• Aquaporins = membrane channels that increase the permeability of certain membranes to water *are where rapid H2O transport is needed in kidneys (H2O needs to be reabsorbed into bloodstream quickly after filtration), secretion of saliva and tears = H2O must flow quickly thru membranes CHAPTER 5 – METHODS FOR STUDYING GENES – MICHEL
• KNOW:

1. role of restriction enzyme (endonuclease) how it works, what it does
2. understand blotting techniques
3. understand how DNA sequencing works
4. understand pathway of nucleotide synthesis
5. understand PCR and why good application for making proteins

• Restriction Enzymes = molecules that cut DNA *recognize palindrome sequences 5’-CCGCGG-3’ bound to 3’-GGCGCC-5’ = 5’ 3’ on both strands are identical *generate blunt cut = cut at symmetry axis or overhang = wind up with 2 strands that have unpaired bases 5’-CCGCGG-3’ cleaved b/w 3 rd^ C and 2 nd^ G bound to 3’-GGCGCC-5’ cleaved b/w 2 nd^ G and 1 st^ C end up w/ 5’-CCGC-3’ bound to 3’-GG-5’ and 3’-CGCC-5’ bound to 5’-GG-3’ – SEE SLIDE *uses = analyze chromosome structure, sequence long DNA molecules, isolate genes, make new DNA molecules that can be cloned

*ex: BamHI b/w G-G, EcoRI b/w G-A, HaeIII at symmetry axis know what happens if given enzyme and DNA sequence and cleave site what the products will be *can use gel electrophoresis w/ multiple restriction enzymes to identify an unknown sequence of DNA b/c you know at what sites the enzymes cleave the DNA use that knowledge to piece sequence togetheronce you get gel

• Southern Blotting = technique used to identify a specific fragment of DNA *steps: 1) run gel and get DNA fragments separated 2) transfer DNA to nitrocellulose membrane 3) add phosphate or fluorescence-labeled DNA probe that will be complementary to the sequence of interest
  4. use autoradiography (X-ray) and reveal probe on an X-ray very similar to Western Blotting
• DNA sequencing Chain-termination method = Sanger dideoxy method *have 4 rxn tubes b/c there are 4 diff bases in DNA *have DNA to be sequenced add primer to begin reaction via DNA poly primer elongated in 4 separate tubes – one containing labeled dATP, dTTP, dCTP, and dGTP along with small [ddNTP] = will add ATP to chain but then rxn will be terminated at that base b/c ddNTP lacks 3’OH after awhile each tube will contain mixtureof prematurely terminated chains at every occurrence of the dNTP (one tube will terminate at As, one at Ts, etc.) new strands are separated and subjected to gel electrophoresis
• Automated DNA Synthesis *1st^ base of interest attached to resin and 2 nd^ base is protected at 5’ sugar end by DMT and at 3’ end by BetaCE undergoes anhydrous coupling and bases are joined by phosphite triester intermediate Oxidation by I 2 changes phosphite into phosphotriester intermediate rinse w/ acid to de-protect 2nd^ base BCE stays b/w 2 bases repeat
• PCR *need: primers that hybridize to flanking sequences of target DNA (10- 30 bp), 4 dNTPs, heat- stable DNA Poly, buffer containing Mg2+ *sequence of events: have target sequence, add excess primers and then heat to separate strands cool to anneal primers heat back up to 72 degrees C and w/ Taq Poly synthesize DNA continue w/ repeating cycles and eventually you can make complements to the complements you have already made b/c you should still have left over primers