MCB 2050 MIDTERM QUESTIONS WITH VERIFIED
ANSWERS
Gene Cloning Steps - Answers - 1) Digest vector and DNA of interest with same RE
2) Anneal/ligate DNA to vectors forming recombinant plasmid
3) Transform plasmid -> E. coli
4) Selection (method of screening) then amplify
Restriction Enzymes (RE) - Answers - - Cut at specific "restriction" sites in DNA
(phosphodiester bond) (palindromic sequences)
Function: degrade invading DNA (don't degrade if methylated)
- Cohesive/blunt ends
3 essential components of cloning, expression, and reporter, plasmids. - Answers - -
origin of replication
-dominant selectable marker (antibiotic resistant gene)
- Unique RE sites for gene insertion
pBluescript - Answers - MCS in lacZ
- if inserted, nonfunctional = white
- if not inserted, functional = blue
Eukaryotic expression vectors - Answers - - Bacterial Ori --> propagate plasmid and
selectable marker
- Eukaryotic promoter, MCS & polyadenylation signal (poly A tail) to express protein of
interest in cell
- Eukaryotic selectable marker, to select cells with plasmid after transfection and drug
selection
Constructing DNA libraries - Answers - - Cut DNA and vector with same RE
- anneal/ligate --> recombinant plasmid
- Plat on agar w/ drug selection
Clone library: each colony has a plasmid with a fragment of DNA, representing a portion
of DNA used to construct the library
Constructing a cDNA library - Answers - -Isolate mRNA and convert to cDNA before
making library
- Reverse Transcription and oligo dT primer = 1st strand
-degrade mRNA w ribonuclease H
- synthesize 2 strand w DNA polymerase (from 1st)
- Screen library by complementation/hybridization
, Complementation - Answers - won't survive without glucose, so remove it to isolate
Hybridization - Answers - radioactive probe complementary to sequence
Electrophoresis - Answers - Size-based separation on gel matrix and electric field
-all = -ve
- shorter = faster
DNA restriction mapping - Answers - DNA map based on size of RE fragments
- comparing different RE's individually and combined
Southern Blot - Answers - Determine presence of specific DNA sequence in genomic
DNA sample using probe hybridized to the sequence
- Cut genomic DNA w/ RE
- Separate fragments on agarose gel
- Transfer DNA fragments to membrane (blot)
- Detect fragments with radioactive prove
uses: detect homologous sequences, disease diagnostics, RFLP analysis (restriction
fragment length polymorphism)
Pros and cons of southern blotting - Answers - pros:
-effective to detect specific DNA in long complex DNA
-quatify amount of DNA present
-Definitive to detect GMO, and cheaper than DNA sequencing
Cons:
-More expensive than most tests
-labor intensive/complex
-time consuming
-requires lot of target DNA
Northern Blotting - Answers - Detect presence, size, abundance of RNA, with a DNA
probe
-RNA separated on agarose gel, rest = same as southern blot
Pro's and cons of northern blotting - Answers - pros:
-relatively fast, low tech, and cheap
Cons:
-less sensitive than PCR (100,000 copies to detect)
Polymerase Chain Reaction (PCR) - Answers - -Forward and reverse primer (amplify
each DNA strand)
Steps: -Denature template DNA (95˚C)
- anneal primers to template (55˚C)
ANSWERS
Gene Cloning Steps - Answers - 1) Digest vector and DNA of interest with same RE
2) Anneal/ligate DNA to vectors forming recombinant plasmid
3) Transform plasmid -> E. coli
4) Selection (method of screening) then amplify
Restriction Enzymes (RE) - Answers - - Cut at specific "restriction" sites in DNA
(phosphodiester bond) (palindromic sequences)
Function: degrade invading DNA (don't degrade if methylated)
- Cohesive/blunt ends
3 essential components of cloning, expression, and reporter, plasmids. - Answers - -
origin of replication
-dominant selectable marker (antibiotic resistant gene)
- Unique RE sites for gene insertion
pBluescript - Answers - MCS in lacZ
- if inserted, nonfunctional = white
- if not inserted, functional = blue
Eukaryotic expression vectors - Answers - - Bacterial Ori --> propagate plasmid and
selectable marker
- Eukaryotic promoter, MCS & polyadenylation signal (poly A tail) to express protein of
interest in cell
- Eukaryotic selectable marker, to select cells with plasmid after transfection and drug
selection
Constructing DNA libraries - Answers - - Cut DNA and vector with same RE
- anneal/ligate --> recombinant plasmid
- Plat on agar w/ drug selection
Clone library: each colony has a plasmid with a fragment of DNA, representing a portion
of DNA used to construct the library
Constructing a cDNA library - Answers - -Isolate mRNA and convert to cDNA before
making library
- Reverse Transcription and oligo dT primer = 1st strand
-degrade mRNA w ribonuclease H
- synthesize 2 strand w DNA polymerase (from 1st)
- Screen library by complementation/hybridization
, Complementation - Answers - won't survive without glucose, so remove it to isolate
Hybridization - Answers - radioactive probe complementary to sequence
Electrophoresis - Answers - Size-based separation on gel matrix and electric field
-all = -ve
- shorter = faster
DNA restriction mapping - Answers - DNA map based on size of RE fragments
- comparing different RE's individually and combined
Southern Blot - Answers - Determine presence of specific DNA sequence in genomic
DNA sample using probe hybridized to the sequence
- Cut genomic DNA w/ RE
- Separate fragments on agarose gel
- Transfer DNA fragments to membrane (blot)
- Detect fragments with radioactive prove
uses: detect homologous sequences, disease diagnostics, RFLP analysis (restriction
fragment length polymorphism)
Pros and cons of southern blotting - Answers - pros:
-effective to detect specific DNA in long complex DNA
-quatify amount of DNA present
-Definitive to detect GMO, and cheaper than DNA sequencing
Cons:
-More expensive than most tests
-labor intensive/complex
-time consuming
-requires lot of target DNA
Northern Blotting - Answers - Detect presence, size, abundance of RNA, with a DNA
probe
-RNA separated on agarose gel, rest = same as southern blot
Pro's and cons of northern blotting - Answers - pros:
-relatively fast, low tech, and cheap
Cons:
-less sensitive than PCR (100,000 copies to detect)
Polymerase Chain Reaction (PCR) - Answers - -Forward and reverse primer (amplify
each DNA strand)
Steps: -Denature template DNA (95˚C)
- anneal primers to template (55˚C)
