MB1-Lecture 1 AGE Online PDF

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A"simple"revision"of"the"major"components"of"macromolecules."The"4"types"or"classes" of"macromolecules."Molecular"Biology"1"will"cover"mostly"nucleic"acids,"but"proteins" (and"enzymes)"will"loom"large"within"many"aspects"of"the"subject."Carbohydrates,"in" parDcular"the"ribose"and"deoxyribose"5-carbon"sugars"will"show"up"as"important"cogs" in"the"structures"of"RNA"and"DNA,"respecDvely."Lipids"will"get"a"menDon"as"they" consDtute"most"of"the"cell"membrane"bilayer"structure." " "

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The"discovery"of"the"structure"of"DNA"in"1953."On"the"leO,"the"original"X-ray"image"of" a"double-stranded"DNA"helix"with"the"even"diameter"depicted."The"dark"shading"top" and"boRom"represents"the"sugar"phosphates"on"the"outside"of"the"double"helix,"and" the"crosses"in"the"centre"are"the"stacked"H-bonded"base"pairs."Right"panel"shows"the" scienDsts"with"their"wire-frame"model."The"analogy"of"a"winding"staircase"is"oOen" cited,"with"the"bannister"rails"represenDng"the"sugar"phosphate"backbone"and"the" steps"as"the"base"pairs"in"the"core"of"the"molecule."An"account"of"the"race"to"discover" the"structure"of"DNA"was"published"by"James"Watson"in"1968,"and"it"makes" compelling"reading."

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The"five"major"bases"in"nucleic"acids."The"purines"have"a"double"ring"with"the"larger" one"being"the"same"as"the"single"ring"in"the"pyrimidine"bases."Thymine"is"only"found" in"DNA."Uracil"is"the"Thymine"equivalent"in"RNA."The"“bases”"are"very"weakly" alkaline,"non-polar,"and"absorb"UV"light"at"a"maximum"wavelength"of"260"nm."

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Four"alphabets."

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The"2"purine"and"3"pyrimidine"bases"that"give"DNA"and"RNA"their"uniqueness"(Uracil" is"only"found"in"RNA;"Thymine"in"DNA)."The"contribuDng"sugars"and"phosphate"are" also"shown."Note"that"DNA"has"deoxyribose"and"RNA"ribose."These"sugars"differ"in" the"2-prime"hydroxyl"posiDon."The"posiDons"are"numbered"according"to"the"sugar" carbon""posiDons"as"indicated"in"the"slide."Deoxyribose"is"really"2-deoxyribose,"but"is" usually"abbreviated"as"‘deoxyribose’."‘Deoxy’"means"‘lacking"oxygen’"and"ribose"is" converted"to"deoxyribose"when"the"2’-carbon"of"ribose"loses"its"hydroxyl"group"(OH),"leaving"a"hydrogen"(H)"in"that"posiDon."" "

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Erwin"Chargaff"performed"experiments"in"the"1930s"that"showed"DNA"to"have"equal" amounts"of"A"and"T;"and"of"G"and"C."These"equaliDes"became"known"as"“Chargaff’s" Rules”."You"can"see"from"the"diagram"how"the"base"pairings"obey"the"rules."Note"that" the"A-T"base"pair"(upper)"has"two"H-bonds"and"the"C-G"pair"three"H-bonds."A"third"Hbond"between"the"H"and"CO"in"the"upper"pair"is"not"formed"due"to"the"distance"being" to"great"for"the"interacDon"to"occur."The"limit"of"an"H-bond"is"about"3.5"angstroms" (Å),"which"is"approximately"three"and"one"half"covalent"bond"distances."Note"for" simplicity,"this"diagram"does"not"show"the"phosphates"that"would"be"connected"to" deoxyribose"on"the"outside.""" "

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The"leO"panel"diagram"shows"how"nucleic"acid"(DNA"or"RNA)"bases"are"paired"by"the" complementarity"rule"(Chargaff"Rules)"and"this"takes"place"across"the"double"helix" and"within"its"hydrophobic"core."The"sugar"phosphate"hydrophilic"“backbone”"of" each"nucleoDde"is"posiDoned"on"the"outside"of"each"helix"strand"facing"the"aqueous" surroundings,"making"the"whole"molecule"soluble"and"acidic."The"small"χαρτοον" (right"panel)"shows"how"the"stacked"base"pairs"gradually"twist"to"give"the"nucleic"acid" its"helical"shape."Each"DNA"or"RNA"nucleoDde"consists"of"base,"sugar,"and"phosphate." Note"the"covalent"aRachment"of"the"nucleoDdes"in"the"verDcal"direcDon"(on"the" outside"of"the"structure)." "

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Base-pairing"in"nucleic"acids."These"pairings"are"always"made,"with"each"larger"purine" base"opposite"a"smaller"pyrimidine"one"so"that"the"DNA"double-stranded"molecule" has"an"even"diameter."The"base-pairing"(dashed"lines)"occurs"due"to"spontaneous" formaDon"of"H-bonds"across"the"gap."Note"the"evenness"of"the"diameter"of"the" paired"bases"that"form"the"double"helix."Note"also"the"opposite'polarity'of"the" strands,"with"each"strand"running"in"opposite"direcDons"(leO"strand"5’"to"3’;"right" strand"3’"to"5’)."This"polarity"is"always"the"same"and"the"double"stranded"nucleic"acid" molecule"will"only"form"and"remain"stable"in"this"opposite"arrangement"of"the" strands."The"5’"designaDon"refers"to"a"free"5’-carbon"on"the"first"nucleoDde"(5’-P"at" the"top"of"the"leO"strand)."The"3’"designaDon"refers"to"a"free"3’-carbon"on"the"last" nucleoDde"of"the"strand"(3’-OH"at"the"boRom"of"the"leO"strand)."Every"nucleoDde"is" joined"covalently"to"the"one"above"and"below"by"phosphodiester'bonds.""" "

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A"double-stranded"‘spiral"staircase’."In"these"views"of"double-stranded"DNA,"note" that"there"are"H-bonds"between"the"nucleoDde"bases"in"the"centre"of"the"molecule" and"it"is"predominantly"these'non-covalent'forces'that'hold'the'two'strands' together."The"outside"of"the"structure"is"very"polar"due"to"the"presence"of"the"sugar" (deoxyribose)"and"phosphates"(negaDvely"charged)"that"surround"the"base"pairs"in" the"centre."DNA"“dissolves”"by"solvaDon"into"water."Note"also"that"the"two"strands" are"anDparallel."The"two"strands"of"DNA"or"RNA"are"held"together"by"the"collecDve" acDon"of"three"types"of"non-"covalent"interacDng"bonds:"(i)"H-bonds"between"the" horizontally"stacked"base"pairs"in"the"core"of"the"molecule;"(ii)"hydrophobic" interacDons"between"the"verDcally"stacked"base"rings"in"the"core;"and"(iii)"sodium" ions"that"neutralise"the"strong"oxygen"negaDve"charges"on"the"acidic"phosphates"on" the"outside"of"the"molecules."Note"in"the"boRom"diagram"that"the"non-polar"bases" are"in"the"centre"and"the"polar"sugar"phosphates"surround"the"non-polar"core"and" are"exposed"to"water.""

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Two"monophosphate"nucleoDdes"joined"with"a"3’"to"5’"phosphodiester"bond"(water' is'eliminated'across'the'bond,'just'as'it'is'in'the'pep:de'bond'between'two'amino' acids)."The"phosphodiester"bond"is"formed"by"the"eliminaDon"of"the"3’-Carbon" hydroxyl"group."These"covalently"joined"nucleoDdes"are"extended"at"either"end"with" new"phosphodiester"bonds"between"added"nucleoDdes,"generaDng"a"polynucleoDde" strand."If"another"anDparallel"strand"is"added,"then"a"double-stranded"DNA"or"RNA" molecule"is"formed."Note"that"the"5-prime"and"3-prime"nomenclature"derives"from" the"sugar"(deoxyribose"in"DNA;"ribose"in"RNA)."Would"the"dinucleoDde"above"be" found"in"DNA"or"RNA?""

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A"simple"representaDon"of"base-pairing"showing"the"precise"(Chargaff"Rules)" complementariDes"of"A"with"T"and"G"with"C"in"any"double-stranded"DNA"molecule."In" RNA,"it"is"A"with"U"(subsDtuDng"for"‘T’)"and"G"with"C."Note"the"anDparallel"(5’"to"3’" and"3’"to"5’)"arrangement"of"the"two"strands."The"5’"ends"carry"free"phosphate" groups"on"the"5’"carbons"of"deoxyribose,"and"3’"ends"carry"free"hydroxyl"groups"on" the"3’"carbons"of"deoxyribose."" "

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We"think"of"a"gene"in"terms"of"the"translated"protein"product,"but"all"genes"have" defined"elements"that"control"the"expression"of"the"gene"(not"shown"here,"but"will" be"illustrated"in"a"later"lecture),"the"iniDaDon"of"transcripDon"(by"the"binding"of"RNA" polymerase"to"the"Promoter"site),"the"ribosome"binding"sequence"that"directs"the" RNA"transcript"to"the"protein"translaDon"site"on"the"ribosome,"and"the"terminaDon" signal"that"enables"RNA"polymerase"to"be"disengaged"from"the"gene"sequence."The" fourth"element"–"the"Open"Reading"Frame"–"is"the"set"of"triplet"nucleoDde"codons" that"specify"the"sequence"of"amino"acids"for"the"translated"polypepDde."

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In"eukaryotes,"the"genes"in"chromosomes"are"arranged"differently"to"those"in" prokaryotes."First"and"foremost"are"the"controlling"elements"or"regions"upstream" (promoter)"and"downstream"(terminaDon"machinery)."The"Open"Reading"Frame" (ORF)"is"non-conDguous"(see"upper"diagram)"and"consists"of"a"series"of"alternate" exons"and"introns."The"exons"are"secDons"of"the"ORF"and"comprise"some"of"the" amino"acid"codons."The"introns,"also"known"as"intervening"sequences,"are"segments" of"innocuous"DNA"that"interrupt"and"act"as"bridges"between"successive"exons."The" mRNA"transcript"made"from"these"genes"comprises"the"enDre"exon-intron"DNA"gene" sequence."This"pre-mRNA"is"then"spliced"within"the"nucleus"to"yield"a"mature"mRNA" transcript"(boRom"of"top"diagram)."This"mRNA"is"now"similar"to"those"from" prokaryotes,"in""that"it"now"comprises"only"the"conjoined,"uninterrupted"exons"with" the"amino"acid"codons"now"conDguous"in"the"manner"of"a"normal"ORF."Note,"all" mRNAs"(from"whichever"source)"do"not"contain"the"promoter"sequence,"which"is" back"upstream"(in"the"5-prime"direcDon)"from"the"ORF."It"is"not"transcribed"because" its"only"purpose"is"in"providing"a"landing"spot"for"RNA"polymerase"to"begin" transcribing"the"gene."The"boRom"diagram"is"an"example"of"a"collagen"gene"with" extensive"exon-intron"structure."We"shall"see"in"a"later"lecture"how"molecular"cloning" gets"around"the"problem"of"eukaryoDc"gene"structure"and"enables"a"copy"of"the"gene" to"be"cloned"and"expressed"in"bacteria"such"as"E.#coli." "

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A"DNA"sequence"begins"and"ends"with"non-coding"nucleoDde"bases"(in"light"grey" type)."An"Open"Reading"Frame"(ORF)"for"a"gene"encodes"a"polypepDde,"which"is" bookended"by"Start"and"Stop"codons"(in"red),"between"which"are"the"triplet"codons" (3"bases"each,"shown"in"blue)"for"the"linear"sequence"(string)"of"amino"acids"that"will" make"up"the"polypepDde"or"protein"for"this"gene."" " One"thing"to"note,"but"which"is"not"shown"here,"is"that"any"changes"in"the"ORF"will" almost"always"be"catastrophic"in"terms"of"the"expression"of"the"protein."DeleDng"or" inserDng"one"or"more"bases"will"alter"the"ORF"and"the"protein"will"not"be"expressed" (translated)."One"or"two"bases"added"or"subtracted"anywhere"will"ruin"the"reading" frame"(the"correct"types"of"amino"acids"in"the"correct"order)."Insert"or"delete"three" bases"and"one"amino"acid"will"be"removed"or"added."While"not"always"deleterious," this"type"of"alteraDon"will"almost"always"lead"to"the"protein"not"folding"correctly,"or" not"working"as"it"should.""" "

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This"cartoon"diagram"contains"an"exaggerated"representaDon"of"circular"plasmid"DNA" molecules"that"are"only"present"in"some"bacteria"and"contain"genes"that"give"an" advantage"to"the"host"bacterium."The"plasmid"is"replicated"synchronously"with"the" chromosome"and"“inherited”"by"daughter"cells"along"with"the"customary"single" chromosome"copy."NaDve"plasmids"may"occur"in"single"to"several"copies"per"cell"and" range"in"size"from"as"small"as"2"kilobases"(kb)"to"perhaps"over"400"kb."The"E.#coli# chromosome"is"4000"kb"in"length"and"very"large"plasmids"may"reach"almost"1/10th" the"size"of"the"chromosome."When"we"come"to"look"at"plasmids"used"in"Molecular" Biology"and"GeneDc"Engineering"(Molecular"Cloning;"Recombinant"DNA"Technology)," we"will"see"that"only"(hybrid)"small"plasmids"are"used"and"these"have"been" engineered"to"contain"special"features"for"molecular"cloning."" "

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Different"views"of"‘supercoils’." "

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DNA"replicaDon"is"a"simple"concept,"but"a"complex"process"involving"a" “choreographic"performance”"by"a"number"of"enzymes"and"enzyme"complexes."In" this"simple"representaDon,"the"parental"DNA"strands"(in"green)"are"relaxed"and" unwound"to"provide"separate"template"strands"for"the"replicaDon"process."In" bacterial"the"mulDsubunit"enzyme"DNA"Polymerase"III"catalyses"the"addiDon"and" linking"of"nucleoDdes"into"new"polynucleoDde"strands"(in"blue)"that"are" complementary"to"the"template"strands"against"which"they"are"copied."""" "

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This"is"the"standard"method"for"idenDfying"and"analysing"nucleic"acids"(DNA,"RNA)."It" is"also"known"as"submarine"gel"electrophoresis"as"the"gel"is"submerged"beneath"the" running"buffer."The"nucleic"acid"samples"are"loaded"in"special"dye"mix"that"contains" marker"dyes"to"indicate"the"extent"of"the"run"–"these"dyes"run"ahead"of" (bromophenol"blue,"BPB)"or"with"(xylene"cyanol)"nucleic"acids"of"various"sizes."The" loading"dyes"also"contain"nuclease"inhibitors"(EDTA)"to"prevent"digesDon"of"DNA" during"the"run,"and"also"contain"a"percentage"of"sucrose"or"a"high"MW"polymer"to" give"the"sample"dye"mix"viscosity"so"that"it"will"seRle"and"remain"in"the"well"unDl"the" run"is"started."Gels"are"run"unDl"the"BPB"dye"reaches"at"least"halfway"or"near"the"end" of"the"gel."Note"that"the"DNA"is"added"at"the"cathode"(negaDve)"end"as"the"DNA"has"a" net"negaDve"charge"at"the"pH"of"the"buffer"(~7.5)"and"will"therefore"be"conducted" towards"the"anode"by"the"negaDvely"charged"buffer"ions"(and"by"their"own"–ve" charges)."The"DNA"molecules"are"conducted"through"the"regularly"sized"pores"of"the" gel."The"migraDon"rates"of"nucleic"acid"molecules"is"determined"by"their"size"and" conformaDon"(see"next"slides).""" "

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The"pore"size"(on"the"right)"is"determined"by"the"agarose"gel"percentage."Most"gels" are"cast"in"the"range"of"0.5"to"3.0%."Higher"gel"percentages"produce"smaller"pore" sizes"and"will"only"allow"ready"migraDon"of"small"fragments."Lower"percentage"gels" will"have"larger"pores"and"allow"larger"nucleic"acids"to"migrate"with"relaDvely"minimal" fricDonal"resistance."In"MB1"All"gels"will"be"around"0.7%."Agarose"is"a"purified" component"of"agar"that"melts"(100oC)"and"sets"(~45oC)"in"the"same"way"as"agar.""

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Loading/running"dye"mixture"is"added"to"DNA"or"RNA"samples"in"aqueous"buffer." Typically,"about"5"µl"dye"to"15"µl"sample."The"loading"dye"mixture"is"supplied"or"made" at"~5x"strength"so"only"a"few"µls"are"required"per"sample."It"contains"buffer,"EDTA," bromophenol"blue"dye"(and"oOen"a"second"dye"xylene"cyanol,"which"is"green"and" runs"slower"than"bromophenol"blue),"and"something"to"make"the"dye"mix"viscous," usually"30%"sucrose"or"a"high"MW"polysaccharide."When"the"sample"is"added"to"the" well"via"a"pipeRe"Dp"(as"in"the"figure"above),"it"drops"into"the"well"as"it"is"more"dense" than"the"running"buffer;"and"it"remains"there"unDl"the"current"is"applied."The"drawing" at"the"boRom"depicts"the"resolving"of"the"DNA"fragment"bands"in"the"gel"as"they" migrate"from"cathode"(-)"to"anode"(+)"(middle"panel)."and"the"completed"run"with"the" fragments"arranged"in"maximum"to"minimum"sizes"from"top"to"boRom."The"gel" photo"at"the"boRom"of"this"slide"depicts"standards"in""the"leO"lane"and"decreasing" amounts"of"the"same"sample"in"lanes"1"to"6."The"point"to"noDce"about"these"samples" is"the"way"the"broadness"of"the"bands"bias"the"actual"size"of"the"DNA"smaple." Although"not"a"precise"issue,"the"actual"size"of"the"sample"band"is"best"taken"from" lane"6."" ""

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Structures"of"the"two"DNA"and"RNA"stains"for"agarose"gels."Ethidium"bromide"(EtBr;" leO)"was"the"original"stain"used"unDl"several"years"ago"(and"sDll"used"in"some"places)." It"has"been"superseded"by"Gel-Red"(right),"which"has"the"same"degree"of"binding"and" staining"sensiDvity"as"EtBr,"but"is"far"less"toxic"and"is"now"widely"used"instead"of"EtBr." You"can"see"from"the"structures"above"that"they"are"comparaDvely"similar,"with"GelRed"ionised"with"Iodine"(I)"versus"bromine(Br)"for"Ethidium."Gel-Red"is"virtually"a" duplicate"of"the"EtBr"mulDple"ring"structure"with"a"long"aliphaDc"connecDng"chain" between"the"rings."Both"dyes"are"planar"(flat)"and"are"able"to"intercalate"(see"next" slides)"between"the"stacked"base"pairs"of"double-stranded"DNA"or"RNA,"or"against" single-stranded"RNA."The"posiDve"charge"on"the"nitrogen"(N+)"allows"the"dye"to"bind" to"the"nucleic"acid’s"opposite"charged"negaDve"phosphates."" ."""""" "

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A"third"type"of"DNA"staining"dye,"which"as"you"can"see"form"the"structure"on"the"leO," will"bind"to"DNA"more"or"less"in"the"same"way"as"Gel-Red"or"ethidium"bromide"(small" posiDve"charge;"intercalaDng"aromaDc"rings)."DNA"stained"with"Sybr"Green"can"be" visualised"by"UV"excitaDon"in"the"same"way"as"the"other"dyes,"though"the"excitaDon" spectrum"is"at"a"higher"wavelength."The"one"property"of"Sybr"Green"that"differs"from" the"other"dyes"is"that"it"does"not"bind"as"well"to"single"stranded"DNA"or"RNA" (fluorescence"is"11x"weaker)."

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DNA"“bands”"in"an"agarose"gel"aOer"electrophoresis"and"stained"with"Sybr"Green"or" Gel-Red"(Irradiate"at"340nm,"visualize"Sybr"Green"at"650nm,"Gel-Red"at"540nm)."The" molecules"of"DNA"(or"RNA)"in"the"original"sample"added"to"the"origin"wells"migrate"in" the"electric"current."They"are"sorted"by"the"current"into"bands"according"to"their" mass"and"conformaDon"so"that"all"molecules"of"the"nucleic"acid"in"any"one"band"are" the"same."The"stained"gel"is"viewed"on"an"ultraviolet"(UV)"light"box"with"the"UV"lamp" set"to"irradiate"the"nucleic"acids"at"a"wavelength"of"302"nm,"whereupon"the"nucleic" acids"absorb"the"UV"light,"fluoresce,"and"re-emit"it"at"540"nm"in"the"red-orange"visible" region"of"the"light"spectrum."Photographic"records"of"the"gel"made"be"made"and" saved"as"jpg"or"Df"files."" "

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A"cartoon"representaDon"of"the"intercala:ng'dye'binding"of"EtBr,"Gel-Red,"or"Sybr" Green"in"double-stranded"DNA."Note"that"the"DNA"“swells”"when"the"dye"is"bound," with"the"enDre"molecule"becoming"less'dense."Dye"binding"is"easier"in"linear"or" relaxed"circular"molecules,"more"difficult"in"supercoiled"DNA"due"to"the"Dghtly"wound" DNA"strands,"making"dye"access"harder."The"planar"rings"“stack”"within"the"core"of" the"structure"like"extra"“stairs”"in"between"the"base"pairs"and"this"stacking"is"aided"by" the"similarity"of"the"hydrophobic"planar"rings"of"the"dyes"to"those"of"the"DNA"or"RNA" bases."""""""""" "

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This"table"gives"approximate"nucleic"acid"separaDon"and"resoluDon"ranges"for"various" percentages"of"agarose"or"polyacrylamide"gels."Note"that"for"smaller"DNA"molecules," gels"must"be"cast"with"polyacrylamide"to"ensure"the"pores"of"the"gel"are"small" enough"to"resolve"the"populaDon"of"DNA"molecules."Agarose"gels"may"be"cast"up"to" 4%"to"resolve"small"DNA"fragments,"but"these"high"%"gels"are"briRle"and"produce"too" much"heat"(by"resistance)"which"could"distort"the"DNA"bands."Note"the"different" posiDonal"profiles"of"the"same"set"of"linear"standards"in"increasing"percentage" agarose"gels"depicted"in"the"gel"photo"on"the"right."This"is"best"illustrated"by"looking" at"the"top"few"standards"in"the"different"%"gels"–"see"how"they"are"spread"out"in"the" 0.7%"gel,"whose"larger"pores"allow"bigger"DNA"molecules"to"migrate"through"the" pores"with"less"fricDonal"resistance."The"1.5%"gel"shows"the"same"high"mass"DNA" bands"“compressed”,"that"is"less"spread"out"and"much"closer"to"the"origin,"because" they"are"too"large"to"migrate"freely"and"encounter"fricDonal"resistance"from"the" smaller"pores."Note"also"that"unlike"circular"nucleic"acid"molecules,"linear"DNA" molecules"migrate"end-on-end,"but"their"lengths"sDll"prevent"them"from"fiwng"easily" through"small"pores"where"their"flexibility"and"length"make"them"bump"conDnuous" into"the"walls"of"the"pores."" "

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EsDmaDng"the"sizes"of"DNA"fragments"typically"involves"running"the"unknown"DNA" against"a"set"of"standards"of"known"sizes."In"the"simple"diagram"above,"it"is"easy"to" make"a"good"guess"of"the"approximate"size"of"the"unknown"fragment,"by"eye."If" nothing"else,"this"at"least"will"tell"you"if"your"more"accurate"values"from"the"standard" curve"are"correct"(as"the"unknown"band"lies"between"two"standards"of"known"sizes)." The"top"lane"has"a"set"of"linear"standards"running"according"to"their"mass/length" sizes"and"will"migrate"according"to"the"inverse"log10"of"their"masses"(see"the"next" slide)."A"DNA"band"of"unknown"size,'but'of'the'same'conforma:on'as'the'set'of' standards"is"run"in"the"boRom"lane."The"hundreds"or"thousands"of"double-stranded" DNA"molecules"in"each"band"are"all"of"the"same"size"and"configuraDon,"which"is"why" they"are"running"at"the"same"posiDon"in"the"gel."Note:"unless"otherwise"stated,"when" reference"is"made"to"circular"or"linear"DNA,"the"molecules"are"deemed"to"be"doublestranded'by"implicaDon,"as"this"is"the"“normal”"state"for"most"such"molecules."Single" stranded"DNA"or"RNA"will"almost"always"be"spelled"ou...