MCB 3020 EXAM 3 STUDY GUIDE
biotechnology - Answers - refers to the use of living organisms to create products that
are useful to humans
Recombinant DNA - Answers - DNA from two or more sources...allows for creation of
DNA sequences not otherwise found in biological organisms
Basic Mechanism of gene cloning - Answers - 1. isolate plasmid DNA from bacterial cell
2. isolate DNA of interest
3. Insert the gene of interest into plasmid DNA...forms recombinant DNA
4. insert the recombinant DNA into a bacterium
5. culture and grow the bacteria
Restriction Enzymes - Answers - a type of endonuclease that cuts up DNA
*normally leave "sticky ends" of DNA
Restrictions sites (recognition sequences) - Answers - short nucleotide sequences that
are recognized by restriction enzymes as the place to cut DNA
normally palindromes
sticky ends - Answers - cut DNA that is left with short, single stranded sequences on
both sides of the fragments
DNA ligase - Answers - seals the two strands of "sticky ended" DNA together by
catalyzing phosphodiester bonds between the single-stranded breaks.
**results in recombinant DNA
Electroporation - Answers - makes a cell competent to pick up DNA from the
environment by applying an electrical shock to it, which increases membrane
permeability.
RecA proteins - Answers - competent cells are engineered to lack these proteins, which
may try to otherwise destroy the inserted sequence or attempt to correct the
recombinant DNA
Reverse Transcriptase - Answers - turns single-stranded RNA into double-stranded
DNA
it is an RNA-dependednt DNA polymerase
Complementary DNA (cDNA) - Answers - the DNA produced by reverse transcriptase
and the RNA template
constructing cDNA - Answers - 1. apply s short poly-T primer to single-stranded RNA
,2. add reverse transcriptase and 4 nucleotides
3. add RNaseH enzyme to cut up the RNA and regenerate the RNA primers
4. add DNA polymerase and DNA ligase to synthesize a new strand
cloning vector - Answers - provides a means for transferring a gene of interest to a host
organism
"good" cloning vectors include - Answers - an origin of replication
a selectable marker
unique restriction sites (polylinkers or multicloning sites)
Plasmid - Answers - *most common cloning vector*
a self-replicating piece of extrachromosomal DNA found in prokaryotes
-mid-sized range (20,000 base pairs or less)
pBR322 and pUC19
bacteriophage - Answers - virus that infects bacteria
carry smaller fragments between 9,000-25,00 base pairs
cosmid - Answers - a hybrid between a plasmid and a phage
carry large fragments between 30,00 and 47,000
artificial chromosomes - Answers - synthetic chromosomes that contain fragments of
DNA integrated into a host chromosome
1. P1 artificial chromosomes (PACs)
2. Bacterial artificial chromosomes (BACs)
3. yeast artificial chromosomes (YACs)
**carry up to 1 MILLION bp
DNA analysis - Answers - used to determine the location of genes, differences in genes,
and where and when genes are expressed
Includes...
1. PCR
2. gel electrophorese
3. restriction fragment analysis
4. blotting techniques
Polymerase chain reaction (PCR) - Answers - quickly and easily makes copies of even
a very small amount of DNA.
targets the DNA with the following:
1. Primers
2. thermostable DNA polymerase (taq polymerase)
3.deoxyribonucleotide triphostates (dATP, dCTP, dGTP, dTTP)
**the solution is heated to 90 to 50 to 70 and then again
, Gel electrophoresis - Answers - a technique that separates nucleic acids and proteins
on the basis of their size and electrical charge, using a gel that is a polymer
then stained with ethidium bromide for DNA to be visible
Restriction fragment length polymorphism (RELP) - Answers - a tool for genetic
fingerprinting, genome mapping, localization of the genes, paternity testing, and
microbial classification.
STR analysis - Answers - compares genomes based on the number of short tandem
repeats
Ribotyping - Answers - variation of RELP that compared ribosomal RNA
Blotting - Answers - technique that combines electrophoresis and hybridization to detect
specific DNA fragments
Southern Blotting - Answers - uses radioactive DNA hybridization probes and
autoradiography to identify the presence or absence of a specific gene
Autoradiography - Answers - method for detecting radioactively labeled molecules
genomic library - Answers - a collection of an organism's entire genomic DNA, which is
stored in a population of identical vectors, each of which has a fragment of the DNA
site-directed mutagenesis - Answers - a technique to introduce a mutation in one or
more of the genes in a genome (target sequence "swaps" for the complementary
sequence that was introduced)
protoplast fusion - Answers - enzymes and other means are used to remove the cell
walls of multiple cells, creating protoplasts. These are then fused together, which is a
large scale method of change to the genome.
**mainly with fungi**
interferons - Answers - the body's "antiviral" molecule
genetic engineering has allowed for the creation of bacteria that specialize in producing
alpha, beta, and gamma interferons
Gene therapy - Answers - the treatment of disease by inserting "therapeutic DNA" into a
patient's cells
hope that specialized transduction can be used for this one day
Primary metabolite - Answers - essential for proper growth, development, and
reproduction
immediate removal of a primary metabolite will impact the microbe's ability to survive
and reproduce
biotechnology - Answers - refers to the use of living organisms to create products that
are useful to humans
Recombinant DNA - Answers - DNA from two or more sources...allows for creation of
DNA sequences not otherwise found in biological organisms
Basic Mechanism of gene cloning - Answers - 1. isolate plasmid DNA from bacterial cell
2. isolate DNA of interest
3. Insert the gene of interest into plasmid DNA...forms recombinant DNA
4. insert the recombinant DNA into a bacterium
5. culture and grow the bacteria
Restriction Enzymes - Answers - a type of endonuclease that cuts up DNA
*normally leave "sticky ends" of DNA
Restrictions sites (recognition sequences) - Answers - short nucleotide sequences that
are recognized by restriction enzymes as the place to cut DNA
normally palindromes
sticky ends - Answers - cut DNA that is left with short, single stranded sequences on
both sides of the fragments
DNA ligase - Answers - seals the two strands of "sticky ended" DNA together by
catalyzing phosphodiester bonds between the single-stranded breaks.
**results in recombinant DNA
Electroporation - Answers - makes a cell competent to pick up DNA from the
environment by applying an electrical shock to it, which increases membrane
permeability.
RecA proteins - Answers - competent cells are engineered to lack these proteins, which
may try to otherwise destroy the inserted sequence or attempt to correct the
recombinant DNA
Reverse Transcriptase - Answers - turns single-stranded RNA into double-stranded
DNA
it is an RNA-dependednt DNA polymerase
Complementary DNA (cDNA) - Answers - the DNA produced by reverse transcriptase
and the RNA template
constructing cDNA - Answers - 1. apply s short poly-T primer to single-stranded RNA
,2. add reverse transcriptase and 4 nucleotides
3. add RNaseH enzyme to cut up the RNA and regenerate the RNA primers
4. add DNA polymerase and DNA ligase to synthesize a new strand
cloning vector - Answers - provides a means for transferring a gene of interest to a host
organism
"good" cloning vectors include - Answers - an origin of replication
a selectable marker
unique restriction sites (polylinkers or multicloning sites)
Plasmid - Answers - *most common cloning vector*
a self-replicating piece of extrachromosomal DNA found in prokaryotes
-mid-sized range (20,000 base pairs or less)
pBR322 and pUC19
bacteriophage - Answers - virus that infects bacteria
carry smaller fragments between 9,000-25,00 base pairs
cosmid - Answers - a hybrid between a plasmid and a phage
carry large fragments between 30,00 and 47,000
artificial chromosomes - Answers - synthetic chromosomes that contain fragments of
DNA integrated into a host chromosome
1. P1 artificial chromosomes (PACs)
2. Bacterial artificial chromosomes (BACs)
3. yeast artificial chromosomes (YACs)
**carry up to 1 MILLION bp
DNA analysis - Answers - used to determine the location of genes, differences in genes,
and where and when genes are expressed
Includes...
1. PCR
2. gel electrophorese
3. restriction fragment analysis
4. blotting techniques
Polymerase chain reaction (PCR) - Answers - quickly and easily makes copies of even
a very small amount of DNA.
targets the DNA with the following:
1. Primers
2. thermostable DNA polymerase (taq polymerase)
3.deoxyribonucleotide triphostates (dATP, dCTP, dGTP, dTTP)
**the solution is heated to 90 to 50 to 70 and then again
, Gel electrophoresis - Answers - a technique that separates nucleic acids and proteins
on the basis of their size and electrical charge, using a gel that is a polymer
then stained with ethidium bromide for DNA to be visible
Restriction fragment length polymorphism (RELP) - Answers - a tool for genetic
fingerprinting, genome mapping, localization of the genes, paternity testing, and
microbial classification.
STR analysis - Answers - compares genomes based on the number of short tandem
repeats
Ribotyping - Answers - variation of RELP that compared ribosomal RNA
Blotting - Answers - technique that combines electrophoresis and hybridization to detect
specific DNA fragments
Southern Blotting - Answers - uses radioactive DNA hybridization probes and
autoradiography to identify the presence or absence of a specific gene
Autoradiography - Answers - method for detecting radioactively labeled molecules
genomic library - Answers - a collection of an organism's entire genomic DNA, which is
stored in a population of identical vectors, each of which has a fragment of the DNA
site-directed mutagenesis - Answers - a technique to introduce a mutation in one or
more of the genes in a genome (target sequence "swaps" for the complementary
sequence that was introduced)
protoplast fusion - Answers - enzymes and other means are used to remove the cell
walls of multiple cells, creating protoplasts. These are then fused together, which is a
large scale method of change to the genome.
**mainly with fungi**
interferons - Answers - the body's "antiviral" molecule
genetic engineering has allowed for the creation of bacteria that specialize in producing
alpha, beta, and gamma interferons
Gene therapy - Answers - the treatment of disease by inserting "therapeutic DNA" into a
patient's cells
hope that specialized transduction can be used for this one day
Primary metabolite - Answers - essential for proper growth, development, and
reproduction
immediate removal of a primary metabolite will impact the microbe's ability to survive
and reproduce
