BCMB 412 - EXAM 1 QUESTIONS WITH
COMPLETE ANSWERS
Be familiar with the experiments determining that DNA is the hereditary material:
Transforming principle in pseudomonas pneumoniae and Hersey and Chase
experiments with phages. - ANSWER - Transforming principles: nonpathogenic (rough)
transformed to pathogenic (smooth) via heat-killed S cells
- Hershey and Chase: viral genes are made of DNA, not proteins -> labeled sulfur and
phosphorous and then examined progeny (phosphorous/DNA still labeled)
Explain how complex organisms such as humans have a relatively small number of
genes compared to other organisms: pleiotropy, alternative splicing and gene
transcription regulation. - ANSWER - pleiotropy: one gene influences more than one
seemingly unrelated phenotype
- alternative splicing: multiple products can be made from one gene
- gene transcription regulation: increasing sophistication results in an increasing
number of tissue-specific products
Understand why model organisms are needed in biological research - ANSWER model
organisms provide insight into biological systems, as fundamental biological principles
are conserved through evolution
What is a good model organism? How is the genome of a good model organism? -
ANSWER short generation time, simple living requirements, small genome with little
repetition, critical mass of researchers, can address important specific problems, able
to be transformed, strongly favored by funding agencies
Be familiar with the basic characteristics and common research applications for the
best characterized model organisms: Phages, E. coli, Yeasts, C. elegans, Drosophila,
mice, and A. Thaliana. - ANSWER - phages: easy transformation, shows how genes can
be turned on/off (lytic vs lysogenic)
- E. coli: optimal model organism, plasmids easy to manipulate, huge populations, single
chromosome
,- yeasts: simple eukaryote, small genome, cell cycle (budding and fission)
- C. elegans: transparent for easy imaging, rapid lifecycle, model for aging process,
human orthologs
- Drosophila: first model organism, multicellular diploid with compact genome, lots of
human disease orthologs
- mice: human disease model, model for development of organ systems
- A. thaliana: plant biology, small genome, easily transformed, fast life cycle
Understand Homologous recombination and how to use it to obtain mouse knockouts -
ANSWER - homologous recombination: genetic information is exchanged by the
crossing over of two similar chromosomes
- knockout: turning off a gene via homologous recombination
Understand the concept of a knockout. - ANSWER add antibiotic resistance & TK
resistance next to gene of interest (TK outside of area of homology), transform ES cells
with DNA via homologous recombination, select for antibiotic resistance, select against
TK via GANC (TK phosphorylates GANC and kills cells that did not undergo homologous
recombination), chimera then bred for complete knockout
Understand the concept of transgene and transgenic animal and the difference between
knockouts and transgenes - ANSWER - transgenic: a new, foreign gene is inserted into
the genome
- knockout: an endogenous gene is turned off/silenced
Recombinant DNA, Restriction enzymes and DNA cloning. cDNA libraries, genomic
libraries and cloning vectors - ANSWER - recombinant DNA: hybridization/DNA from
multiple sources (ex. insulin reproduced by recombinant DNA in bacteria)
- restriction enzymes and DNA cloning: restriction enzyme digests plasmid/vector
(antibiotic marker & restriction site) and gene of interest, gene of interest is integrated
into plasmid & sealed with ligase, plasmid amplified
- cDNA libraries: collection of DNA clones in vectors that represent all genes expressed
in a genome (cDNA = DNA without introns, obtained via RT)
- genomic libraries: collection of DNA clones in vectors that represent full genome
, (doesn't guarantee expression due to presence of introns)
- cloning vector: plasmid with a restriction site and antibiotic resistance marker
DNA gel electrophoresis - ANSWER a procedure used to separate DNA fragments
according to their size; shorter fragments travel farther (small surface area, able to
travel more easily through agarose gel)
SDS PAGE electrophoresis (PAGE: Poly acrylamide gel electrophoresis) - ANSWER SDS
gel electrophoresis separates proteins by size, SDS-PAGE = Western blot (detects
presence/concentration of proteins via presence of antibodies)
PCR. Understand the basic principle and components of a PCR reaction. How the DNA
is amplified. What is the role of temperature changes in every step of each cycle? Why
do we need to repeat cycles multiple times? - ANSWER - denaturation: high
temperatures denature strands of DNA, leaving them open
- annealing: temperature lowered and primers are put into place
- extension: temperature raised and thermostable Taq polymerase extends chains using
free dNTPs in the solution
- exponential amplification
Reverse Transcriptase-PCR. (RT-PCR) - ANSWER PCR technique that involves
converting RNA to DNA by reverse transcriptase; used to make cDNA libraries
Chromatography columns - ANSWER - ion exchange: agarose beads with negative
charge will attract negatively charged proteins and repel positively charged proteins ->
determines charge
- gel-filtration: large proteins are unable to enter agarose beads, smaller proteins will
enter first at the top -> determines size
Affinity Chromatography columns - ANSWER coupling agarose beads with specific
reagents that interact with protein of interest to facilitate purification -> works to purify
specifically-identified proteins
COMPLETE ANSWERS
Be familiar with the experiments determining that DNA is the hereditary material:
Transforming principle in pseudomonas pneumoniae and Hersey and Chase
experiments with phages. - ANSWER - Transforming principles: nonpathogenic (rough)
transformed to pathogenic (smooth) via heat-killed S cells
- Hershey and Chase: viral genes are made of DNA, not proteins -> labeled sulfur and
phosphorous and then examined progeny (phosphorous/DNA still labeled)
Explain how complex organisms such as humans have a relatively small number of
genes compared to other organisms: pleiotropy, alternative splicing and gene
transcription regulation. - ANSWER - pleiotropy: one gene influences more than one
seemingly unrelated phenotype
- alternative splicing: multiple products can be made from one gene
- gene transcription regulation: increasing sophistication results in an increasing
number of tissue-specific products
Understand why model organisms are needed in biological research - ANSWER model
organisms provide insight into biological systems, as fundamental biological principles
are conserved through evolution
What is a good model organism? How is the genome of a good model organism? -
ANSWER short generation time, simple living requirements, small genome with little
repetition, critical mass of researchers, can address important specific problems, able
to be transformed, strongly favored by funding agencies
Be familiar with the basic characteristics and common research applications for the
best characterized model organisms: Phages, E. coli, Yeasts, C. elegans, Drosophila,
mice, and A. Thaliana. - ANSWER - phages: easy transformation, shows how genes can
be turned on/off (lytic vs lysogenic)
- E. coli: optimal model organism, plasmids easy to manipulate, huge populations, single
chromosome
,- yeasts: simple eukaryote, small genome, cell cycle (budding and fission)
- C. elegans: transparent for easy imaging, rapid lifecycle, model for aging process,
human orthologs
- Drosophila: first model organism, multicellular diploid with compact genome, lots of
human disease orthologs
- mice: human disease model, model for development of organ systems
- A. thaliana: plant biology, small genome, easily transformed, fast life cycle
Understand Homologous recombination and how to use it to obtain mouse knockouts -
ANSWER - homologous recombination: genetic information is exchanged by the
crossing over of two similar chromosomes
- knockout: turning off a gene via homologous recombination
Understand the concept of a knockout. - ANSWER add antibiotic resistance & TK
resistance next to gene of interest (TK outside of area of homology), transform ES cells
with DNA via homologous recombination, select for antibiotic resistance, select against
TK via GANC (TK phosphorylates GANC and kills cells that did not undergo homologous
recombination), chimera then bred for complete knockout
Understand the concept of transgene and transgenic animal and the difference between
knockouts and transgenes - ANSWER - transgenic: a new, foreign gene is inserted into
the genome
- knockout: an endogenous gene is turned off/silenced
Recombinant DNA, Restriction enzymes and DNA cloning. cDNA libraries, genomic
libraries and cloning vectors - ANSWER - recombinant DNA: hybridization/DNA from
multiple sources (ex. insulin reproduced by recombinant DNA in bacteria)
- restriction enzymes and DNA cloning: restriction enzyme digests plasmid/vector
(antibiotic marker & restriction site) and gene of interest, gene of interest is integrated
into plasmid & sealed with ligase, plasmid amplified
- cDNA libraries: collection of DNA clones in vectors that represent all genes expressed
in a genome (cDNA = DNA without introns, obtained via RT)
- genomic libraries: collection of DNA clones in vectors that represent full genome
, (doesn't guarantee expression due to presence of introns)
- cloning vector: plasmid with a restriction site and antibiotic resistance marker
DNA gel electrophoresis - ANSWER a procedure used to separate DNA fragments
according to their size; shorter fragments travel farther (small surface area, able to
travel more easily through agarose gel)
SDS PAGE electrophoresis (PAGE: Poly acrylamide gel electrophoresis) - ANSWER SDS
gel electrophoresis separates proteins by size, SDS-PAGE = Western blot (detects
presence/concentration of proteins via presence of antibodies)
PCR. Understand the basic principle and components of a PCR reaction. How the DNA
is amplified. What is the role of temperature changes in every step of each cycle? Why
do we need to repeat cycles multiple times? - ANSWER - denaturation: high
temperatures denature strands of DNA, leaving them open
- annealing: temperature lowered and primers are put into place
- extension: temperature raised and thermostable Taq polymerase extends chains using
free dNTPs in the solution
- exponential amplification
Reverse Transcriptase-PCR. (RT-PCR) - ANSWER PCR technique that involves
converting RNA to DNA by reverse transcriptase; used to make cDNA libraries
Chromatography columns - ANSWER - ion exchange: agarose beads with negative
charge will attract negatively charged proteins and repel positively charged proteins ->
determines charge
- gel-filtration: large proteins are unable to enter agarose beads, smaller proteins will
enter first at the top -> determines size
Affinity Chromatography columns - ANSWER coupling agarose beads with specific
reagents that interact with protein of interest to facilitate purification -> works to purify
specifically-identified proteins
