BIOL 142 Lecture Notes - Lecture 18: Agarose Gel Electrophoresis, Bamhi, Antimicrobial Resistance
30 Apr 2018
School
Department
Course
Professor
!"#$%&'$"()'*
+'#"%,-"("%,"%)#.*'$"/%010
2
3..4%'$%-.,%567%8(.9"#%'("%:#"/
2
0.,%("#$();$).<%"<=>&"#%,.(4
2
?+@
2
7##.;)'$).<%#$:/)"#
0.,%>.:%;.:*/%)/"<$)A>%'%B"<"%,)$-%'**"*"#% $-'$%;':#"#%'%/)#"'#"
○
?"/)B(""%'<'*>#"#%#-.,)<B%)A%'%8-"<.$>8"%)#%-"()$'9*"%
0.,%;'<%>.:%)/"<$)A>%B"<"%;':#)<B%$-)#
§
@C3?%'<'*>#)#
§
○
2
1.#D-'$%)#%("8("#"<$"/%9>%EAmpRF%'</%EOriF%)<%$-"%8G+%8*'#&)/%9"*.,H
ANSWERI
Ori:#origin#of#replication
6""/"/%A.(%;"**%$.%4<.,%$.%;.8>%.:(%(";.&9)<'<$%8*'#&)/
○
2
AmpR:#antibiotic#(ampicillin)#resistance
7&8);)**)<%)#%'<%'<$)9).$);
○
2
C*:%B"<"%J-.,%$.%;*.<"%)$%)<$.%8*'#&)/H%G#)<B%)$%'#%'%K";$.(%$.%9"%
+:$%,)$-%L'&0M%K)'%#$);4>%"</#
3..4%'$%B)K"<%("#$();$).<%"</.<:;*"'#"%'</%:#"%.<"%$.%#:9N;*.<"%$-"%
B"<"%)<$.%$-"%8*'#&)/
§
○
!('<#A.(&%9';$"()'*%;"**#% 9>%8)8"$$)<B%.:(%567%A(.&%.:(%*)B'$).<%("';$).<%
O)<#"($).<P%)<$.%'%K)'*%.A%;.&8"$"<$%9';$"()'*%;"**#Q%A.**.,%8(.$.;.*%A.(%
$('<#A.(&'$).<Q%$-"<%"R8.#"%$.%;"**#
D'<$%$-"&%$.%;.8>%*.$#%.A%;.8)"#%.A%.:(%(";.&9)<'<$%'("'%.A%#$:/>
§
○
2
6"R$%#$"8I%8*'$)<B%;"**#%,)$-%'<$)9).$);%("#)#$'<;"%#.%.<*>%9';$"()'%,)$-%$-"%8*'#&)/%,)**%
B(.,
0'#%(";.&9)<'<$%8*'#&)/%.A%)<$"("#$2
D"%/.<S$%;'("%'9.:$%;"**#%,)$-.:$%$-"%8*'#&)/%J<""/%.<"#%,)$-%)$%$.%;.8>%'</%
#$:/>
2
T&8.($'<$%$.%)/"<$)A>%$-"%;"**%$-'$%-'#%$-"%8*'#&)/%'</%;.8>)<B%$-"%8*'#&)/2
2. 7%;)(;:*'(%8*'#&)/%)#%UVWW%98%)<%*"<B$-%'</%-'#%(";.B<)$).<%#"X:"<;"#%'$%$-"%
A.**.,)<B%*.;'$).<#I%UWWY%ZWWY%MUWWY%MZWWY%'</%UWWW[ !-)#%8*'#&)/%)#%;.&8*"$"*>%
/)B"#$"/%'</%$-"%A('B&"<$#%'("%#"8'('$"/%9>%B"*%"*";$(.8-.("#)#[ 0.,%&'<>%9'</#%
,)**%9"%K)#:'*)="/%.<%$-"%'B'(.#"%B"*H
U7[
3B.
\+[
]5[
Z^[
EXPLANATION
+)(;:*'(%8*'#&)/2
!.*/%;:$%#)$"#%'("%'$%$-"%8.#)$).<#%'9.K"2
D-"<%;:$$)<B%$-"%;)(;:*'(% $-)<B%'$%$-.#"%#)$"#Q%>.:%("*"'#"%A('B&"<$#%.A%'%
/)AA"("<$%#)="
C('B&"<$#%.A%#'&"%#)="%'("%)<%$-"%#'&"%8*';"
○
UVWW%NUWWW%_UWW%`%MWWW%%98
UWW%98I%UWW
§
○
\WW%98
ZWW%NUWW%`%\WW%98
§
MZWW%NMUWW%`%\WW%98
§
UWWW%NMZWW%`%\WW%98
§
○
ZWW%98
MUWW%NZWW%`%ZWW%9?
§
○
2
3. The maps of the sites for restriction enzyme R in wild type and mutated
Alzheimer’s patients are shown. Assume this is a recessive disease. Samples of
DNA from children (c) and their parents (F and M) were analyzed by gel
electrophoresis followed by Southern blot using a radioactively labeled probe
shown in the figure. The pattern observed on the X-ray film is shown. Which
children will develop the disease?
ANSWERI%C1#and#C3
!.%-'K"%7*=-")&"(S#Q%>.:%&:#$%-'K"%U%;.8)"#
+*.#"%$.%$-"%#-.($%A.(&#%.A%&'(4"(
○
U%,-.%.<*>%-'K"%$-"%#-.($%A.(&%.A%$-"%&'(4"(%
○
2
a:$'<$%-'#%8("#"<;"%.A%'<.$-"(%;:$%#)$"
a:$'<$%)#%*)<4"/%$.%$-"%#-.($%B"<"
○
T<%$-"%,)*/%$>8"Q%)$%,)**%-'K"%'%*.<B"(%B"<"
○
2
]%49%)#%*'(B"(%A('B&"<$Q%b%492
4. The following cross is made between two plants:
AaBb
X
aabb
.The F1
offspring occur in the following numbers: 399
Aabb
, 401
aaBb
, 98
AaBb
,
102
aabb
.
Which of the following illustrates the allelic arrangement of the
A
and
B
genes in
cells that undergo meiosis in the
AaBb
parent?
ANSWERI%E
U%8.##)9)*)$)"#
5.&)<'<$%'**"*"#% '("%.<%$-"%#'&"%-.&.*.B%'</%(";"##)K"%'("%.<%$-"%.$-"(
AABB'</%aabb ,)$-%<.%(";.&9)<'$).<%"K"<%<""/"/
§
7[
5.&)<'<$%'**"*"#% '("%)<%$('<#L[
2
bcc_\WM_cV_MWU`MQWWW%
7'99%\Wd
○
''L9%\Wd
○
7'L9%MWd
○
''99%MWd
○
2
U%8.)<$%$"#$%;(.##%&'$)<B%'%/.:9*"%-"$"(.=>B.:#%,)$-%#.&".<"%-.&.=>B.:#%
(";"##)K"
TA%:<*)<4"/Q%,"%"R8";$%"X:'*%8(.8.($).<#
D.:*/%"R8";$%.AA#8()<B%$.%9"%AaBb '</%aabbQ%9:$%$-">%-'88"<%*"##%
.A$"<%-"("Q%'**.,)<B%:#%$.%"R;*:/"%7
§
○
e<"#%$-'$%A.(&%'$%*.,"(%A("X:"<;>%'("%(";.&9)<'<$#
○
e:(%&.#$%<:&"(.:#%.<"#%'("%Aabb '</%aaBb
@";.&9)<'$).<%.<*>%.;;:(#%UWd%.A%$-"%$)&"
§
○
2
5. T<%$-"%8()&:*'%8*'<$Q%A*.,"(%;.*.(%)#%$-"%("#:*$%.A%"8)#$'#)#[% !-"%8)B&"<$%&'*K)/)<%
O
M
P%;("'$"#%9*:"%A*.,"(#[ TA%$-)#%B"<"%)#%-.&.=>B.:#%(";"##)K"Q%$-"%A*.,"(%)#%<.$%
9*:"[ 7*#.Q%8(./:;$).<%.A%&'*K)/)<%;'<%9"%#:88("##"/%)A%$-"("%)#%'%/.&)<'<$%'**"*"%'$%
B"<"
D
[TA%$,.%8*'<$#%-"$"(.=>B.:#%A.(%9.$-%B"<"#%'("%;(.##"/Q%,-'$%,)**%9"%$-"%
8-"<.$>8);%('$).%.A%9*:"%$.%<.<9*:"%A*.,"(#H
ANSWERI%13#not#blue:#3#blue
1"<.$>8"%Jf8-"<.$>8"2
ag%`%9*:"%A*.,"(2
&&%`%<.$%9*:"2
5g%`%#:88("##%9*:"2
//%`%<.%'AA";$%.<%9*:"2
MmDd R%MmDd
c%ag5g%Jf%<.$%9*:"
○
b%ag//%Jf%9*:"
○
b%&&5g%<.$%9*:"
○
M%&&//%Jf%<.$%9*:"
○
2
7. !-"%&'8%/)#$'<;"%9"$,""<%B"<"#%'%'</%9%)#%;'*;:*'$"/%'#%V[b%&:%A(.&%$-"%A.**.,)<B%
;(.##I%♂7'L9%h%♀''99[% D-);-%.A%$-"%A.**.,)<B%#$'$"&"<$#%'9.:$%$-)#%;(.##%)#%;.((";$H
8.3%#of#the#gametes#from#the#male#parent#are#recombinantA.
cM[id%.A%$-"%B'&"#%A(.&%$-"%&'*"%8'("<$%'("%(";.&9)<'<$L[
V[bd%.A%$-"%B'&"$"#%A(.&%$-"%A"&'*"%8'("<$%'("%(";.&9)<'<$+[
cM[id%.A%$-"%B'&"#%A(.&%$-"%A"&'*"%8'("<$%'("%(";.&9)<'<$5[
EXPLANATIONI
a'*"%-'#%K'()'9)*)$>%)<%'**"*"#Q%.<*>%8*';"%,-"("%,"%;'<%#""%(";.&9)<'$).<%
"K"<$#%)<%$-"%;(.##
2
+'<%.<*>%/"$"(&)<"%&:%:#)<B%'%UN8.)<$%$"#$%;(.##%'88(.';-2
Exam%2%Review%Day
D"/<"#/'>Q% a'(;-%UVQ%UWMV
MUIWM%?a
!"#$%&'$"()'*
+'#"%,-"("%,"%)#.*'$"/%0102
3..4%'$%-.,%567%8(.9"#%'("%:#"/2
0.,%("#$();$).<%"<=>&"#%,.(42
?+@2
7##.;)'$).<%#$:/)"#
0.,%>.:%;.:*/%)/"<$)A>%'%B"<"%,)$-%'**"*"#% $-'$%;':#"#%'%/)#"'#"
○
?"/)B(""%'<'*>#"#%#-.,)<B%)A%'%8-"<.$>8"%)#%-"()$'9*"%
0.,%;'<%>.:%)/"<$)A>%B"<"%;':#)<B%$-)#
§
@C3?%'<'*>#)#
§
○
2
1.#D-'$%)#%("8("#"<$"/%9>%EAmpRF%'</%EOriF%)<%$-"%8G+%8*'#&)/%9"*.,H
ANSWERI
Ori:#origin#of#replication
6""/"/%A.(%;"**%$.%4<.,%$.%;.8>%.:(%(";.&9)<'<$%8*'#&)/
○
2
AmpR:#antibiotic#(ampicillin)#resistance
7&8);)**)<%)#%'<%'<$)9).$);
○
2
C*:%B"<"%J-.,%$.%;*.<"%)$%)<$.%8*'#&)/H%G#)<B%)$%'#%'%K";$.(%$.%9"%
+:$%,)$-%L'&0M%K)'%#$);4>%"</#
3..4%'$%B)K"<%("#$();$).<%"</.<:;*"'#"%'</%:#"%.<"%$.%#:9N;*.<"%$-"%
B"<"%)<$.%$-"%8*'#&)/
§
○
!('<#A.(&%9';$"()'*%;"**#% 9>%8)8"$$)<B%.:(%567%A(.&%.:(%*)B'$).<%("';$).<%
O)<#"($).<P%)<$.%'%K)'*%.A%;.&8"$"<$%9';$"()'*%;"**#Q%A.**.,%8(.$.;.*%A.(%
$('<#A.(&'$).<Q%$-"<%"R8.#"%$.%;"**#
D'<$%$-"&%$.%;.8>%*.$#%.A%;.8)"#%.A%.:(%(";.&9)<'<$%'("'%.A%#$:/>
§
○
2
6"R$%#$"8I%8*'$)<B%;"**#%,)$-%'<$)9).$);%("#)#$'<;"%#.%.<*>%9';$"()'%,)$-%$-"%8*'#&)/%,)**%
B(.,
0'#%(";.&9)<'<$%8*'#&)/%.A%)<$"("#$
2
D"%/.<S$%;'("%'9.:$%;"**#%,)$-.:$%$-"%8*'#&)/%J<""/%.<"#%,)$-%)$%$.%;.8>%'</%
#$:/>
2
T&8.($'<$%$.%)/"<$)A>%$-"%;"**%$-'$%-'#%$-"%8*'#&)/%'</%;.8>)<B%$-"%8*'#&)/
2
2. 7%;)(;:*'(%8*'#&)/%)#%UVWW%98%)<%*"<B$-%'</%-'#%(";.B<)$).<%#"X:"<;"#%'$%$-"%
A.**.,)<B%*.;'$).<#I%UWWY%ZWWY%MUWWY%MZWWY%'</%UWWW[ !-)#%8*'#&)/%)#%;.&8*"$"*>%
/)B"#$"/%'</%$-"%A('B&"<$#%'("%#"8'('$"/%9>%B"*%"*";$(.8-.("#)#[ 0.,%&'<>%9'</#%
,)**%9"%K)#:'*)="/%.<%$-"%'B'(.#"%B"*H
U
7[
3
B.
\
+[
]
5[
Z
^[
EXPLANATION
+)(;:*'(%8*'#&)/
2
!.*/%;:$%#)$"#%'("%'$%$-"%8.#)$).<#%'9.K"
2
D-"<%;:$$)<B%$-"%;)(;:*'(% $-)<B%'$%$-.#"%#)$"#Q%>.:%("*"'#"%A('B&"<$#%.A%'%
/)AA"("<$%#)="
C('B&"<$#%.A%#'&"%#)="%'("%)<%$-"%#'&"%8*';"
○
UVWW%NUWWW%_UWW%`%MWWW%%98
UWW%98I%UWW
§
○
\WW%98
ZWW%NUWW%`%\WW%98
§
MZWW%NMUWW%`%\WW%98
§
UWWW%NMZWW%`%\WW%98
§
○
ZWW%98
MUWW%NZWW%`%ZWW%9?
§
○
2
3. The maps of the sites for restriction enzyme R in wild type and mutated
Alzheimer’s patients are shown. Assume this is a recessive disease. Samples of
DNA from children (c) and their parents (F and M) were analyzed by gel
electrophoresis followed by Southern blot using a radioactively labeled probe
shown in the figure. The pattern observed on the X-ray film is shown. Which
children will develop the disease?
ANSWERI%C1#and#C3
!.%-'K"%7*=-")&"(S#Q%>.:%&:#$%-'K"%U%;.8)"#
+*.#"%$.%$-"%#-.($%A.(&#%.A%&'(4"(
○
U%,-.%.<*>%-'K"%$-"%#-.($%A.(&%.A%$-"%&'(4"(%
○
2
a:$'<$%-'#%8("#"<;"%.A%'<.$-"(%;:$%#)$"
a:$'<$%)#%*)<4"/%$.%$-"%#-.($%B"<"
○
T<%$-"%,)*/%$>8"Q%)$%,)**%-'K"%'%*.<B"(%B"<"
○
2
]%49%)#%*'(B"(%A('B&"<$Q%b%492
4. The following cross is made between two plants:
AaBb
X
aabb
.The F1
offspring occur in the following numbers: 399
Aabb
, 401
aaBb
, 98
AaBb
,
102
aabb
.
Which of the following illustrates the allelic arrangement of the
A
and
B
genes in
cells that undergo meiosis in the
AaBb
parent?
ANSWERI%E
U%8.##)9)*)$)"#
5.&)<'<$%'**"*"#% '("%.<%$-"%#'&"%-.&.*.B%'</%(";"##)K"%'("%.<%$-"%.$-"(
AABB'</%aabb ,)$-%<.%(";.&9)<'$).<%"K"<%<""/"/
§
7[
5.&)<'<$%'**"*"#% '("%)<%$('<#L[
2
bcc_\WM_cV_MWU`MQWWW%
7'99%\Wd
○
''L9%\Wd
○
7'L9%MWd
○
''99%MWd
○
2
U%8.)<$%$"#$%;(.##%&'$)<B%'%/.:9*"%-"$"(.=>B.:#%,)$-%#.&".<"%-.&.=>B.:#%
(";"##)K"
TA%:<*)<4"/Q%,"%"R8";$%"X:'*%8(.8.($).<#
D.:*/%"R8";$%.AA#8()<B%$.%9"%AaBb '</%aabbQ%9:$%$-">%-'88"<%*"##%
.A$"<%-"("Q%'**.,)<B%:#%$.%"R;*:/"%7
§
○
e<"#%$-'$%A.(&%'$%*.,"(%A("X:"<;>%'("%(";.&9)<'<$#
○
e:(%&.#$%<:&"(.:#%.<"#%'("%Aabb '</%aaBb
@";.&9)<'$).<%.<*>%.;;:(#%UWd%.A%$-"%$)&"
§
○
2
5. T<%$-"%8()&:*'%8*'<$Q%A*.,"(%;.*.(%)#%$-"%("#:*$%.A%"8)#$'#)#[% !-"%8)B&"<$%&'*K)/)<%
O
M
P%;("'$"#%9*:"%A*.,"(#[ TA%$-)#%B"<"%)#%-.&.=>B.:#%(";"##)K"Q%$-"%A*.,"(%)#%<.$%
9*:"[ 7*#.Q%8(./:;$).<%.A%&'*K)/)<%;'<%9"%#:88("##"/%)A%$-"("%)#%'%/.&)<'<$%'**"*"%'$%
B"<"
D
[TA%$,.%8*'<$#%-"$"(.=>B.:#%A.(%9.$-%B"<"#%'("%;(.##"/Q%,-'$%,)**%9"%$-"%
8-"<.$>8);%('$).%.A%9*:"%$.%<.<9*:"%A*.,"(#H
ANSWERI%13#not#blue:#3#blue
1"<.$>8"%Jf8-"<.$>8"2
ag%`%9*:"%A*.,"(2
&&%`%<.$%9*:"2
5g%`%#:88("##%9*:"2
//%`%<.%'AA";$%.<%9*:"2
MmDd R%MmDd
c%ag5g%Jf%<.$%9*:"
○
b%ag//%Jf%9*:"
○
b%&&5g%<.$%9*:"
○
M%&&//%Jf%<.$%9*:"
○
2
7. !-"%&'8%/)#$'<;"%9"$,""<%B"<"#%'%'</%9%)#%;'*;:*'$"/%'#%V[b%&:%A(.&%$-"%A.**.,)<B%
;(.##I%♂7'L9%h%♀''99[% D-);-%.A%$-"%A.**.,)<B%#$'$"&"<$#%'9.:$%$-)#%;(.##%)#%;.((";$H
8.3%#of#the#gametes#from#the#male#parent#are#recombinantA.
cM[id%.A%$-"%B'&"#%A(.&%$-"%&'*"%8'("<$%'("%(";.&9)<'<$L[
V[bd%.A%$-"%B'&"$"#%A(.&%$-"%A"&'*"%8'("<$%'("%(";.&9)<'<$+[
cM[id%.A%$-"%B'&"#%A(.&%$-"%A"&'*"%8'("<$%'("%(";.&9)<'<$5[
EXPLANATIONI
a'*"%-'#%K'()'9)*)$>%)<%'**"*"#Q%.<*>%8*';"%,-"("%,"%;'<%#""%(";.&9)<'$).<%
"K"<$#%)<%$-"%;(.##
2
+'<%.<*>%/"$"(&)<"%&:%:#)<B%'%UN8.)<$%$"#$%;(.##%'88(.';-2
Exam%2%Review%Day
D"/<"#/'>Q% a'(;-%UVQ%UWMV MUIWM%?a
Document Summary
How you could identify a gene with alleles that causes a disease. Pedigree analyses showing if a phenotype is heritable. Needed for cell to know to copy our recombinant plasmid. Look at given restriction endonuclease and use one to sub-clone the gene into the plasmid. Transform bacterial cells by pipetting our dna from our ligation reaction (insertion) into a vial of competent bacterial cells, follow protocol for transformation, then expose to cells transformation, then expose to cells. Want them to copy lots of copies of our recombinant area of study. Next step: plating cells with antibiotic resistance so only bacteria with the plasmid will grow. We don"t care about cells without the plasmid need ones with it to copy and study. Important to identify the cell that has the plasmid and copying the plasmid: a circular plasmid is 2800 bp in length and has recognition sequences at the following locations: 200; 600; 1200; 1600; and 2000.
