2017 Midterm answers - practice test PDF

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Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' BIOLOGY'309' Midterm'Examination' Monday'June'5,'2017' ' ' ' This'examination'is'out'of'46'marks'+'2'bonus'marks.' ' You'have'50'minutes'to'complete'the'examination.' ' This'examination'counts'for'25%'of'your'final'mark.'' ' ' ' Instructions:'' • Remember'to'read'all'questions'carefully'and'completely'before'answering.'' • Write'in'pen'if'you'want'your'paper'to'be'considered'for'remarking.'' • No'additional'materials'are'allowed'–'no'calculators'or'other'devices.'' • Write'you'answers'in'the'space'provided.'' • Provide'answers'in'enough'detail'so'we'can'understand'your'logic.'If'answers'are'vague,'we' cannot'give'you'full'marks.'' ' • Academic'integrity'guidance:'Make'every'reasonable'effort'to'prevent'other'students' around'you'from'seeing'your'answers.'Similarly,'keep'your'eyes'on'your'own'paper.'' ' ' ' ' '

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Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' ' 1. Clearly'distinguish'between'the'following'(1'mark'each)' a. Endonuclease'and'Exonuclease' Endonucleases'cut'/cleave'DNA'sequences'into'two'different'pieces'internally'while' exonucleases'cuts'or'degrades'DNA'sequences'at'their'ends.'' ' b. Transcriptional'fusion'construct'and'Translational'fusion'construct' Transcriptional'fusion'constructs'test'the'ability'of'a'promoter'to'turn'on'the'transcription'(and' translation)'of'a'reporter'gene'that'is'located'downstream'of'the'promoter.'Translational'fusion' constructs'are'designed'to'fuse'the'ORF'of'a'gene'with'a'reporter'gene'so'that'the' localization/movement'of'the'protein'product'can'be'studied' ' In'general,'this'question'was'not'answered'well'-'but'I'am'not'sure'why'because'we'had'a'whole' tutorial'on'it.'' ' c. Screen'and'Selection' Screen'is'to'detect'the'presence'or'absence'of'a'mutant/desired'phenotype'(think'of'blue/white' screening'or'screening'a'library'with'a'probe'for'a'positive'clone)' ' Selection'is'to'select'for'a'mutant/desired'phenotype/trait'that'has'a'growth'advantage'under' certain'conditions'(E.g.'selection'of'transformed'host'cells'containing'a'vector'with'a'antibiotic' resistance'gene'by'growing'the'transformed'cells'in'LB'broth'containing'that'antibiotic)' ' You'did'not'need'to'give'examples'to'get'the'mark'–'I'am'simply'providing'them'to'help'with' your'understanding'of'the'concept.'' ' d. Primer'and'Probe' A'primer'is'a'short'oligonucleotide'that'binds'to'a'complementary'template'nucleotide' sequence'and'is'required'for'initiation'of'in#vitro'DNA'synthesis'reactions.' ' A'probe'is'a'labeled'nucleotide'sequence'(can'be'short'or'long,'either'DNA'or'RNA)'that'is'used' to'screen'a'library'to'identify'clones'possessing'inserts'with'complementary'sequence'' ' 2. Give'one'example'of'a'trait'of'a'model'organism'and'briefly'explain'when'this'trait'would'be' advantageous'for'studies'in'molecular'biology'(2'marks)' One'of'the'following'traits'and'a'reason'why'that'trait'aids'the'study'of'molecular'biology'(this' question'was'on'page'46'of'your'course'notes)' Small'genome'size,'low'amount'of'repetitive'DNA,'diploid,'short'life'cycle,'small'+'cheap'to' maintain'(note,'the'reason'behind'why'this'is'a'selected'trait'is'because'it'allows'the'researcher'to' use'more'organisms'per'experiment'and'thus'increase'the'n'number'and'the'validity'of'their' experiments),'or'transformable' ' 3. Give'an'example'of'when'you'would'want'to'generate'a'genomic'library'using'partially'digested' genomic'DNA'versus'completely'digested'genomic'DNA.'(1'mark)''' 1'mark'for'one'of'the'following:' -Find'adjacent'regions'(promoters,'enhancers,'other'genes)'to'your'gene'of'interest'–'could'give' any'example'of'this.''

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Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' -For'use'in'helping'to'assemble'a'whole'genome'(overlapping'ends'help'piece'the'fragments' together)' ' Gave'0.5'marks'for'finding'other'gene'family'members'as'long'as'it'was'clear'that'you'were' suggesting'that'the'gene'family'members'were'close'by'your'gene'of'interest.'' ' ' ' 4. Match'the'task'from'Column'2'with'the'methods'listed'in'Column'1'by'placing'the'task'number' in'the'empty'box'beside'each'method.'Each'method'can'only'be'used'once.'(10'marks)' ' Column'1:'Method' Column'2:'Task' ' 4' Oligo'dT'affinity' 1.''Synthesis'of'radiolabeled'probes'for'screening'a' chromatography' cDNA'library'to'identify'exon-intron'splice'sites' 10' Centrifugation' 2.''Detection'of'the'number'of'amplicons'generated' in'an'in#vitro'DNA'synthesis'reaction' Restriction'digest' 3.''Concentration'of'plasmid'DNA' 6' 8'

In#silico'analysis'

9'

Electroporation'

5'

Heat'shock'

1'

Polymerase'chain'reaction'

3'

Ethanol'precipitation'

2'

Electrophoresis'

7'

Spectrometry'

4.''Purification'of'mRNA'from'other'nucleic'acids'in'a' cleared'homogenate'derived'from'cells'or'tissues' 5.''Introduce'a'purified'plasmid'into'a'bacterial'host' cell'' 6.''Linearization'of'a'plasmid'for'generation'of'a' riboprobe' 7.''Quantification'of'the'concentration'of'DNA'in'a' sample' 8.''Prediction'of'restriction'enzyme'cut'sites'in'a' known'DNA'sequence' 9.''Creation'of'a'DNA'library'with'high' transformation'efficiency' 10.'Removal'of'growth'media'from'E.#coli'in'order'to' resuspend'cells'in'different'buffer''

' 5. In'the'following'table,'indicate'whether'the'statements'apply'to'the'creation,'screening'and/or' use'of'a'genomic'library'or'cDNA'library'by'placing'an'“X”'in'the'appropriate'column.'If'the' statement'does'not'apply'to'either'type'of'library,'put'“N/A”'for'“not'applicable”.''(5'marks)' ' Genomic'library' cDNA'library' ' (Eukaryote)' (Eukaryote)' Restriction'enzymes'are'needed'to'generate' X' X'(will'also'accept)' the'library'(this'was'referring'to'making'the' DNA'fragments'but'I'think'some'students' also'thought'it'was'referring'to'the'vector,' so'I'have'accepted'two'answers'here'–'if'you' were'thinking'of'linearizing'the'vector'with' restriction'enzymes,'then'it'should'apply'to' both'the'genomic'and'cDNA'library.'If'you' were'thinking'of'creating'the'fragments,' Page 3 of 10'

Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' then'this'would'just'apply'to'the'genomic' library.'If'you'said'it'only'applied'to'the' cDNA'library,'then'you'did'not'get'the'mark.'' Too'big'so'need'a' X'(average'size'of' Fragments'are'cloned'into'the'commonly' replacement'vector,' insert'is'2000'bp)' used'plasmid'vector' cosmid'vector'or'artificial' chromosome' Cloned'fragments'contain'intron'sequences' X' Intron'sequences'' are'spliced'from' pre-mRNA' transcript,'so'mRNA' will'not'contain' intron'sequences' and'will'not'be' present'in'the' library' Can'provide'insight'into'the'cellular' DNA'does'not'change'in' X'(recall'how'we' cells'in'response'to' talked'about'gene' response'to'changes'in'environment'(e.g.' environment' expression'–'mRnA' temperature'change)' transcripts-'chaging' in'response'to' environment)' Can'be'used'to'screen'for'protein'products' Fragments'in'the'clones' X' will'contain'introns'–'thus' will'interrupt'ORF'and'no' protein'can'be'produced' ' ' '

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Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' ' 6. Describe'briefly'why'each'of'the'following'steps'is'important:'(6'marks'–'1'mark'each)' a. Inclusion'of'a'negative'control'during'restriction'digests' ' To'ensure'the'plasmid'DNA'was'not'degraded'by'contaminating'nucleases'(the'negative'control' would'be'plasmid'DNA'and'buffer'but'no'restriction'enzyme,'incubated'for'the'same' time/temperature'as'the'test'sample)'' ' b. Inserts'for'cosmid'vectors'must'be'32-45'kb.''' Inserts'must'be'of'a'minimum'size'to'ensure'the'phage'DNA'is'large'enough'to'fill'the'head'of'the' phage'and'ensure'capsid'head'stability.'If'the'insert'is'too'large,'the'DNA'genome'will'be'too'big'to' fit'into'the'phage'head.'' ' c. Inclusion'of'the'lambda'endonuclease'in'the'in#vitro'lambda'phage'packaging'extracts' ' The'lambda'endonuclease'is'important'to'cleave'the'concatamer'DNA'or'cosmid'DNA'at'the'cos' site'to'generate'a'linear'genome.'The'endonuclease'is'also'associated'with'the'phage'head'and' helps'to'stuff'the'linearize'genome'into'the'phage'head'' ' d. The'inclusion'of'0.3'M'sodium'acetate'with'2.5'volumes'of'ethanol'during'DNA'precipitation' ' The'positively'charged'sodium'ions'in'the'sodium'acetate'interacts'with'the'negatively'charged' DNA'backbone'and'allows'the'strands'of'DNA'to'come'close'together'because'the'electrostatic' hinderance'is'minimized.'This'helps'to'precipitate'the'DNA'in'the'presence'of'ethanol' ' ' e. To'use'70%'ethanol'for'washing'the'DNA'pellet'during'DNA'isolation' The'use'of'70%'ethanol'allows'excess'salt'ions'to'be'removed'from'the'DNA'pellet'because'of'the' 30%'water'content'while'still'keeping'the'DNA'precipitated'because'of'the'high'%'of'ethanol.'' ' ' f. The'inclusion'of'EDTA'in'nucleic'acid'extraction'buffers' ' EDTA'chelates'Mg2+'ions'–'Mg2+'ions'are'important'co-factors'for'many'enzymes'including' nucleases'and'the'chelation'of'the'Mg2+'ions'causes'the'inactivation'of'the'enzymes.'Thus,'this' step'helps'to'maintain'the'integrity'of'your'sample.'' ' ' Bonus'Question'1:'Which'of'the'following'enzymes'does'not'require'a'primer'to'initiate'nucleic' acid'synthesis?''(circle'your'answer;'1'mark)' ' DNA'polymerase' ' RNA'polymerase' ' Reverse'transcriptase' ' Bonus'Question'2:'Which'of'the'following'three'probe'labeling'methods'results'in'the'generation' of'the'least'sensitive'probe'–'i.e.'is'not'easily'detectable?'(circle'your'answer;'1'mark)' ' DNA'synthesis'via'PCR' ' Riboprobes'via'in'vitro'RNA'synthesis' Klenow'fill-in''

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Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' 7. You'set'up'a'PCR'mix'as'follows:' a. In'the'below'list,'fill'in'the'blanks'to'indicate'what'solvent'you'should'use,'the'volume'of'the' solvent'you'should'add'and'the'total'volume'of'your'reaction.''Include'units.'(3'marks)' ' Component' ' ' ' ' ' ' ' Volume' 10x'buffer'containing'salts'and'MgCl2' ' ' ' 5'µL' 10x'stock'of'dNTP'mixture' ' ' ' ' ' 1'µL' Sense'primer' ' ' ' ' ' ' ' 1'µL' Antisense'primer' ' ' ' ' ' ' 1'µL' Taq'polymerase' ' ' ' ' ' ' 0.5'µL' ' ____41.5'µL_________?' Solvent_______sterile'nuclease'free'water'________?' ' ' Total'volume' ' ' ' ' ' ' ' _____50'µL______?' For'the'solvent:' You'got'1'mark'for'the'following'answers:' -Ultrapure'water' -Nuclease'free'water' -Sterile'nuclease'free'water'(this'is'the'best'answer)' ' You'got'0.5'mark'if'you'said:' -water' -DI'water'or'distilled'water' -nuclease'free' ' For'the'calculation,'if'you'got'one'of'the'numbers'incorrect'you'only'lost'one'mark'as'long'as'the' rest'of'your'calculation'was'correct.'(i.e.'you'were'not'double'penalized)' ' b. List'two'important'criteria'for'designing'primers'to'ensure'they'result'in'amplification'of'your' region'of'interest'during'a'PCR'reaction.'(2'marks)' They'should'be'complementary'and'specific'to'the'DNA'region'you'are'trying'to'amplify' They'should'both'have'similar'annealing'temperatures'–within'2-3'degrees'of'each'other' They'should'not'be'self'complementary'or'complementary'to'the'other'primer' They'should'have'~50%'GC'content'to'ensure'the'primer'annealing'temperature'is'>40%'and' helps'to'increase'specificity' ' ' c. The'sequence'of'your'sense'primer'is'5’-ATGGTGTAAGGCCACTTGC-3’'and'the'sequence'of' your'antisense'primer'is'5’-TCTAGCCAGTCATGAGTAGA-3’.'Based'on'these'primers,'what' annealing'temperature'would'you'choose'to'use'in'the'thermocycling'protocol?'Briefly'explain' in'one'(1)'sentence'why'you'chose'that'annealing'temperature'(2'marks).'' ' The'sense'primer'and'antisense'primer'both'have'a'Tm'of'58.'Thus,'ideally'you'should'have' said'you'would'want'the'annealing'temperature'of'your'primers'to'be'slightly'lower'than'58-' remember'that'the'Tm'is'the'temperature'at'which'half'of'the'double'stranded'DNA'is' denatured'(see'page'221'of'your'course'notes).'We'accepted'Tms'in'the'range'of'54-58'so'that' the'primers'would'bind'to'the'template'DNA'and'yet'be'at'a'high'enough'temperature'to' prevent'non-specific'binding.'' ' Page 6 of 10'

Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' We'gave'part'marks'for'the'following:' -if'you'had'the'formula'for'calculating'the'Tm:'0.5'mark' -if'you'calculated'the'Tm'correctly,'but'no'rationale:'1'mark' -if'you'calculated'the'Tm'wrong'but'provided'good'rationale:'1'mark' -If'you'calculated'the'Tm'to'be'58'for'one'of'the'primers'but'not'the'other,'and'had'good' rationale:'1.75'marks.' ' ' d. If'you'wanted'to'create'a'labeled'probe,'how'would'you'modify'the'components'of'your'PCR' mix?'Be'specific.'(1'mark)' Use'a'mixture'of'dNTPs'that'has'one'of'the'dNTPs'either'radiolabeled'or'DIG'labeled:'' dATP,'dTTP,'dGTP32,'dCTP'(you'could'have'chosen'to'radiolabel'any'dNTP)' OR:'dATP,'dTTP,'dGTP-DIG,'dCTP'(you'could'have'chosen'to'DIG'label'any'dNTP)' ' If'you'said'use'radiolabeled'(or'DIG-labeled)'dNTPs,'you'got'0.5'marks.'Only'one'of'the'dNTPs' should'be'labeled.'' ' Also,'I'did'not'take'away'marks'for'this,'but'I'want'students'to'be'careful'about'this'in'the' future'–'some'students'indicated'the'labeled'dNTP'would'be'dUTP.'But'remember'–'this'is'an' in'vitro'synthesis'reaction'and'thus'there'is'no'uracil'here'(and'it'would'be'written'as'UTP' because'it'is'not'a'deoxynucleotide)' ' ' e. Assuming'that'your'primer'pair'is'valid'and'specific'to'your'target,'that'you'have'correctly' selected'your'thermocycling'conditions'and'your'PCR'machine'is'working,'do'you'think'you' will'be'able'to'amplify'your'fragment'of'interest'using'the'PCR'mix'that'you'made'up'in'Part'A' of'this'question?'Why'or'why'not?'(2'marks)' ' No'–'the'PCR'mix'from'part'A'is'missing'the'DNA'template.'Without'the'DNA'template,'the' reaction'will'not'work'as'a'template'is'essential'for'an'in#vitro'DNA'synthesis'reaction'to'occur.'' ' '

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Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' ' 8. What'went'wrong?'For'each'of'the'following'scenarios,'identify'what'went'wrong.'' ' a. Using'PCR,'you'generated'a'radiolabelled'probe'to'the'promoter'region'for'your'gene'of' interest.'You'use'it'to'probe'a'cDNA'library.'After'exposing'the'membranes'to'film,'you'are' unable'to'detect'any'positive'clones.'Why'did'your'hybridization'experiment'not'yield'any' positive'clones?'(1'mark)' ' cDNA'libraries'do'not'contain'promoter'regions'-'remember,'cDNA'is'a'complementary'DNA' strand'to'the'mRNA'isolated'from'a'cell'and'mRNA'transcripts'do'not'have'promoter'regions.' Thus,'there'are'no'clones'in'the'library'for'the'probe'to'bind'to'and'thus'there'will'not'be'any' positive'clones.'' ' I'gave'0.5'marks'for'the'following'because'it'demonstrated'that'you'were'thinking'of'all'the' ways'a'hybridization'could'go'wrong:' -did'not'denature'cDNA' -failed'to'add'probe' -not'enough'time'for'probe'to'hybridize' -cDNA'does'not'contain'complementary'sequence' -stringency'was'too'high' -DNA'had'degraded'while'making'the'library' -used'the'wrong'probe'(although'this'one'was'a'bit'of'stretch)' -a'cDNA'library'is'a'snapshot'of'a'tissue'at'a'certain'point'of'time'and'thus'your'transcript' might'not'be'expressed'and'thus'represented'in'the'library'(this'would'have'been'an' OUTSTANDING'answer'had'it'not'been'that'the'researcher'was'searching'for'a'promoter' region'in'a'cDNA'library)' ' b. After'plasmid'ligation'and'transformation'by'electroporation,'your'agar'selection'plate' contains'only'blue'colonies.'List'two'possible'reasons'for'this'result.''(2'marks)' ' -You'forgot'to'add'the'insert'to'the'ligation'reaction'and'plasmid'has'self-ligated'and'does'not' contain'an'insert.'' -Insert'was'ligated'into'the'vector'but'was'small'and'did'not'interrupt'the'LacZ’'gene'reading' frame'and'thus'the'LacZ'protein'was'produced'by'alpha'complementation'once'transformed' into'the'host'cell'(contains'the'other'half'of'the'LacZ'gene)'and'thus'colonies'appear'blue' ' 0.5'mark'was'given'for'“insert'ligated'outside'of'the'LacZ’'gene' ' c. After'screening'your'genomic'DNA'library'with'a'homologous'probe,'you'obtain'hundreds'of' positive'clones.'List'two'possible'reasons'for'this'result'and'how'you'could'fix'each'(2'marks)' ' 1'mark'for'any'of'the'following:'' -Stringency'of'was'too'low'–'you'could'increase'the'stringency'of'your'wash'by'decreasing'salt' concentrations'and/or'increasing'the'temperature'to'remove'non-specific'binding'of'the'probe'to' clones.'' -Probe'is'detecting'a'gene'family'and'their'sequences'are'highly'similar.'Redesign'your'probe'so' that'it'is'complementary'to'a'region'that'is'unique'amongst'members'of'the'gene'family.' -Probe'was'too'short'–'not'specific'enough.'Design'longer'probe' Page 8 of 10'

Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' -Probe'was'designed'to'a'repetitive'sequence.'Redesign'probe'to'a'region'with'low'repetitive' sequence/unique'sequence.' ' d. You'ligate'a'fragment'into'a'plasmid'containing'an'ampicillin'resistance'gene'and'use'heat' shock'to'transform'the'E.#coli,'followed'by'recovery'in'LB'+'ampicillin'broth.'You'plate'some'of' the'transformation'mix'on'your'selection'plate'and'grow'overnight.'The'following'day,'there' are'no'colonies'on'the'agar'plate.'Why?'(1'mark)' ' Broth'contains'ampicillin'and'thus'the'newly'transformed'cells'will'not'have'had'enough'time'to' transcribe'and'translate'the'ampicillin'resistance'gene'encoded'on'the'plasmid'(we'discussed'this' together'in'class).'' ' Half'mark'if'you'said'one'of'the'following:' -Cells'were'not'competent' -Heat'shocked'your'cells'for'too'long' -plated'cells'on'the'wrong'selection'plate'(e.g.'agar'plate'contained'kanamycin'instead'of' ampicillin)' -improper'incubation'temperature/conditions' -time'in'recovery''was'not'long'enough' ' e. You'quantify'a'sample'of'plasmid'DNA'on'the'nanodrop'by'measuring'the'OD260'nm'and'obtain' a'value'of'100'µg/mL.'However,'when'you'quantify'the'same'sample'of'plasmid'DNA'using'gel' electrophoresis'and'a'mass'ruler,'you'obtain'a'value'of'60'µg/mL.'List'two'reasons'why'there' is'a'discrepancy'between'the'DNA'concentrations'obtained'via'the'two'methods'used'to' quantify'the'same'sample'of'plasmid'DNA.'(2'marks)' ' 1'mark'for'any'of'the'following'(need'two'for'the'answer):'' -Sample'could'be'contaminated'with'phenol'–'would'artificially'inflate'the'OD260nm'reading' -Sample'could'be'contaminated'with'protein'–'would'artificially'inflate'the'OD260nm'reading' -The'mass'ruler'gives'an'estimate'of'DNA'concentration'–based'off'of'visualization'of'band' intensity'on'the'gel'and'not'an'absolute'quantification.'' -Some'of'the'DNA'has'degraded'–'still'quantified'by'nanodrop'but'would'not'be'present'in'the' band'of'interest'on'the'gel'' -there'were'multiple'fragments'in'the'sample'(e.g.'nicked'circles,'supercoiled'plasmid,'linear' plasmid)'which'would'all'be'quantified'by'the'nanodrop'but'perhaps'you'were'only'estimating'the' amount'of'DNA'in'one'of'the'bands'using'the'mass'ruler'and'electrophoresis.'' -you'forgot'to'zero'the'nanodrop'using'the'solvent'alone'and'thus'the'solvent'that'the'plasmid' DNA'is'in'is'artificially'inflating'your'absorbance'reading.'' -ethidium'bromide'does'not'bind'to'supercoiled'plasmid'DNA'in'as'high'of'a'level'as'nonsupercoiled'DNA'and'thus'this'would'impact'your'ability'to'effectively'quantify'the'amount'of'DNA' present'using'a'mass'ruler'(based'off'of'intensity'of'the'bands)' ' ' ' ' --------------------End'of'Midterm'Evaluation----------------------' Marks'will'not'be'given'for'anything'written'below.'' ' Page 9 of 10'

Name:______________________________________________________'''''''''''''''''''''''''''ID:_____________________________' ' ' ' Question' Marks' Grade' 1' 4' ' 2' 2' ' 3' 1' ' 4' 10' ' 5' 5' ' 6' 6' ' 7' 10' ' 8' 8'' ' Bonus' 2' ' Total' 46+2' ' ...