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3. You plan to construct a mouse genomic DNA library, using the plasmid pUC19 shown below as cloning vector. The mouse DNA has already been purified, and digested with the restriction enzyme BamHI. EcoRI KpnI BamHI Sall HincII AccII PstI HindIII SacI XmaI SmaI XbaI BspMI SphI Restriction sites lacZ* ori pUC19 ampr a) How does the construction of a genomic DNA library differ from that of a cDNA library? b) Give one advantage and one disadvantage of having constructed a mouse genomic library (rather than a cDNA library). c) You have constructed the mouse genomic library by cloning into the BamHI site of pUC19, and transformation into E. coli. Explain how you would use a combination of antibiotic and colour selection to identify colonies containing potential genomic clones or recombinant plasmids. d) You want to select a clone from your genomic library that contains gene A. It is known that gene A encodes protein A, with .... His – Cys – Met – Ser – Arg – Phe ..... as part of its amino acid sequence. You wish to make a set of 18 bp DNA probes to screen your DNA library with. i) Considering the degeneracy of the genetic code, how many possible different DNA sequences could encode this amino acid sequence? ii) Give the DNA sequence of any one probe that would be suitable to use. iii) You find that this probe you have designed does not detect any colonies in the genomic library, however it successfully binds to colonies from a cDNA library prepared from the same mouse. Give a possible explanation for this observation. Second base U C A G U UUU Phe UCU Ser UAU Tyr UGU Cys U UUC UCC UAC UGC C UUA Leu UCA UAA Stop UGA Stop A UUG UCG UAG Stop UGG Trp G C CUU Leu CCU Pro CAU His CGU Arg U CUC CCC CAC CGC C CUA CCA CAA Gln CGA A CUG CCG CAG CGG G A AUU Ile ACU Thr AAU Asn AGU Ser U AUC ACC AAC AGC C AUA ACA AAA Lys AGA Arg A AUG Met ACG AAG AGG G G GUU Val GCU Ala GAU Asp GGU Gly U GUC GCC GAC GGC C GUA GCA GAA Glu GGA A GUG GCG GAG GGG G

3. You plan to construct a mouse genomic DNA library, using the plasmid pUC19 shown below as cloning vector. The mouse DNA has already been purified, and digested with the restriction enzyme BamHI.

EcoRI KpnI BamHI Sall HincII AccII PstI HindIII
SacI XmaI SmaI XbaI BspMI SphI
Restriction sites
lacZ*
ori
pUC19
ampr

a) How does the construction of a genomic DNA library differ from that of a cDNA library?
b) Give one advantage and one disadvantage of having constructed a mouse genomic library (rather than a cDNA library).
c) You have constructed the mouse genomic library by cloning into the BamHI site of pUC19, and transformation into E. coli. Explain how you would use a combination of antibiotic and colour selection to identify colonies containing potential genomic clones or recombinant plasmids.
d) You want to select a clone from your genomic library that contains gene A. It is known that gene A encodes protein A, with .... His – Cys – Met – Ser – Arg – Phe ..... as part of its amino acid sequence. You wish to make a set of 18 bp DNA probes to screen your DNA library with.
i) Considering the degeneracy of the genetic code, how many possible different DNA sequences could encode this amino acid sequence?
ii) Give the DNA sequence of any one probe that would be suitable to use.
iii) You find that this probe you have designed does not detect any colonies in the genomic library, however it successfully binds to colonies from a cDNA library prepared from the same mouse. Give a possible explanation for this observation.

Second base
U C A G
U UUU Phe UCU Ser UAU Tyr UGU Cys U
UUC UCC UAC UGC C
UUA Leu UCA UAA Stop UGA Stop A
UUG UCG UAG Stop UGG Trp G
C CUU Leu CCU Pro CAU His CGU Arg U
CUC CCC CAC CGC C
CUA CCA CAA Gln CGA A
CUG CCG CAG CGG G
A AUU Ile ACU Thr AAU Asn AGU Ser U
AUC ACC AAC AGC C
AUA ACA AAA Lys AGA Arg A
AUG Met ACG AAG AGG G
G GUU Val GCU Ala GAU Asp GGU Gly U
GUC GCC GAC GGC C
GUA GCA GAA Glu GGA A
GUG GCG GAG GGG G

3. You plan to construct a mouse genomic DNA library, using the plasmid pUC19 shown below as cloning vector. The mouse DNA has already been purified, and digested with the restriction enzyme BamHI.

EcoRI KpnI BamHI Sall HincII AccII PstI HindIII
SacI XmaI SmaI XbaI BspMI SphI
Restriction sites
lacZ*
ori
pUC19
ampr

a) How does the construction of a genomic DNA library differ from that of a cDNA library?
b) Give one advantage and one disadvantage of having constructed a mouse genomic library (rather than a cDNA library).
c) You have constructed the mouse genomic library by cloning into the BamHI site of pUC19, and transformation into E. coli. Explain how you would use a combination of antibiotic and colour selection to identify colonies containing potential genomic clones or recombinant plasmids.
d) You want to select a clone from your genomic library that contains gene A. It is known that gene A encodes protein A, with .... His – Cys – Met – Ser – Arg – Phe ..... as part of its amino acid sequence. You wish to make a set of 18 bp DNA probes to screen your DNA library with.
i) Considering the degeneracy of the genetic code, how many possible different DNA sequences could encode this amino acid sequence?
ii) Give the DNA sequence of any one probe that would be suitable to use.
iii) You find that this probe you have designed does not detect any colonies in the genomic library, however it successfully binds to colonies from a cDNA library prepared from the same mouse. Give a possible explanation for this observation.

Second base
U C A G
U UUU Phe UCU Ser UAU Tyr UGU Cys U
UUC UCC UAC UGC C
UUA Leu UCA UAA Stop UGA Stop A
UUG UCG UAG Stop UGG Trp G
C CUU Leu CCU Pro CAU His CGU Arg U
CUC CCC CAC CGC C
CUA CCA CAA Gln CGA A
CUG CCG CAG CGG G
A AUU Ile ACU Thr AAU Asn AGU Ser U
AUC ACC AAC AGC C
AUA ACA AAA Lys AGA Arg A
AUG Met ACG AAG AGG G
G GUU Val GCU Ala GAU Asp GGU Gly U
GUC GCC GAC GGC C
GUA GCA GAA Glu GGA A
GUG GCG GAG GGG G

Added by Sarah H.

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