BIOL 301 Exam 3 | Actual study set |
Questions and verified Answers
Plasmid - ANSW-- in bacterial cells
- extra chromosomal DNA
- small, circular, double-stranded
- some have multiple copies of itself within the cell
3 Common Properties of Cloning Vectors - ANSW-- replicate autonomously
- many have antibiotic-resistance genes
- unique restriction site facilitates cloning of insert DNA
Bacteriophage Vector - ANSW-- constructed from phage chromosome
- central ⅓ of chromosome has genes required for lysogeny but not lytic growth. This part gets excised
- foreign DNA is inserted in the 10-15 kb slot
Cosmid Vector - ANSW-- hybrid between plasmid and phage chromosome
- replicate autonomously in E. Coli
- can be packaged in phage heads
- accepts inserts of 35-45 kb
Phagemid Vector - ANSW-- contains components from plasmids and phage chromosome
- replicate in E. Coli as double stranded DNA
- helper phage can turn on phage mode, which is where single-stranded DNA will be packaged in phage
head
Blue/ White Sceening - ANSW-- The E. coli lacZ gene encodes beta-galactosidase
- beta-galactosidase converts colorless Xgal substrate into a blue product
- blue colonies contain beta-galactosidase and white colonies do not
Cloning your gene - ANSW-1. plasmid is cleaved with enzyme to cut at the site that you want
2. DNA with gene you want is digested with same enzyme
3. Put linearized plasmid DNA with foreign DNA fragments so cohesive ends will bind
4. Recombinants will form (some with gene that you want and some with gene that you desired)
Amplification of cloned gene occurs... (2 ways) - ANSW-1. Vector gets replicated by host cell many times
(lots of copies per cell)
2. Bacterial cell divides approximately every 20 minutes (millions of cells overnight)
Side note: Telomeres, centromeres and other highly repetitive sequences can be cloned as well
Shuttle Vector - ANSW-Are able to replicate in E. coli as well as mammals
YAC (Yeast Artificial Chromosome) - ANSW-- Genetically engineered yeast minichromosome
- Accept foreign DNA segments from 200-500 kb
, Composition: Yeast OriC, yeast centromere, 2 yeast telomeres, a selectable marker, and a polycloning
site
BAC (bacterial artificial chromosome) - ANSW-- Constructed from bacterial fertility factors
- accept foreign DNA segments from 150-300 kb
- less complex than YAC
PAC (bacteriophage artificial chromosome) - ANSW-- Constructed from bacteriophage P1 chromosomes
- accept foreign DNA segments from 150-300 kb
- less complex than YAC
genomic DNA library - ANSW-Set of DNA clones collectively containing the entire genome of an organism
cDNA library - ANSW-contains the coding regions of the expressed genes of an organism. It is made of
complementary DNA (cDNA) synthesized from RNA by reverse transcriptase.
Genetic Selection - ANSW-searching for a DNA sequence that restores the wild-type phenotype to a
mutant organism
Molecular Hybridization - ANSW-Molecular hybridization is based on the hybridization of similar DNA
sequences
Cloning Vector - ANSW-- a small piece of DNA that can be stably maintained in an organism, and into
which a foreign DNA fragment can be inserted for cloning purposes.
- used to obtain numerous copies of inserted DNA segment
- can be plasmids, cosmids, phages, BACs, YACs, or MACs
- comprise of an OriC, unique restriction site, and selectable marker
Expression Vector - ANSW-- a plasmid that is used to introduce a specific, foreign gene into a target cell
and carry out expression of the gene by commandeering the molecular machinery of the target cell
- used to obtain gene product of DNA insert (either RNA or protein)
- this is a plasmid vector
- comprises of enhancers, termination codon, transcription initiation sequence, and translation initiation
sequence along with all of the components of a typical cloning vector
Southern blotting - ANSW-Molecule Detected: DNA
Gel Electroph: Agarose
Gel Pretreatment: Depurination, denaturation, and neutralization
Blotting method: capillary transfer
Probes: DNA radioactive or nonradioactive
Northern blotting - ANSW-Molecule Detected: mRNA
Gel Electrop: Formaldehyde agarose
Gel Pretreatment: None
Blotting Method: Capillary transfer
Probes: cDNA, cRNA radioactive or nonradioactive
Western blotting - ANSW-Molecule Detected: Protein
Gel electroph: polyacrylamide
Questions and verified Answers
Plasmid - ANSW-- in bacterial cells
- extra chromosomal DNA
- small, circular, double-stranded
- some have multiple copies of itself within the cell
3 Common Properties of Cloning Vectors - ANSW-- replicate autonomously
- many have antibiotic-resistance genes
- unique restriction site facilitates cloning of insert DNA
Bacteriophage Vector - ANSW-- constructed from phage chromosome
- central ⅓ of chromosome has genes required for lysogeny but not lytic growth. This part gets excised
- foreign DNA is inserted in the 10-15 kb slot
Cosmid Vector - ANSW-- hybrid between plasmid and phage chromosome
- replicate autonomously in E. Coli
- can be packaged in phage heads
- accepts inserts of 35-45 kb
Phagemid Vector - ANSW-- contains components from plasmids and phage chromosome
- replicate in E. Coli as double stranded DNA
- helper phage can turn on phage mode, which is where single-stranded DNA will be packaged in phage
head
Blue/ White Sceening - ANSW-- The E. coli lacZ gene encodes beta-galactosidase
- beta-galactosidase converts colorless Xgal substrate into a blue product
- blue colonies contain beta-galactosidase and white colonies do not
Cloning your gene - ANSW-1. plasmid is cleaved with enzyme to cut at the site that you want
2. DNA with gene you want is digested with same enzyme
3. Put linearized plasmid DNA with foreign DNA fragments so cohesive ends will bind
4. Recombinants will form (some with gene that you want and some with gene that you desired)
Amplification of cloned gene occurs... (2 ways) - ANSW-1. Vector gets replicated by host cell many times
(lots of copies per cell)
2. Bacterial cell divides approximately every 20 minutes (millions of cells overnight)
Side note: Telomeres, centromeres and other highly repetitive sequences can be cloned as well
Shuttle Vector - ANSW-Are able to replicate in E. coli as well as mammals
YAC (Yeast Artificial Chromosome) - ANSW-- Genetically engineered yeast minichromosome
- Accept foreign DNA segments from 200-500 kb
, Composition: Yeast OriC, yeast centromere, 2 yeast telomeres, a selectable marker, and a polycloning
site
BAC (bacterial artificial chromosome) - ANSW-- Constructed from bacterial fertility factors
- accept foreign DNA segments from 150-300 kb
- less complex than YAC
PAC (bacteriophage artificial chromosome) - ANSW-- Constructed from bacteriophage P1 chromosomes
- accept foreign DNA segments from 150-300 kb
- less complex than YAC
genomic DNA library - ANSW-Set of DNA clones collectively containing the entire genome of an organism
cDNA library - ANSW-contains the coding regions of the expressed genes of an organism. It is made of
complementary DNA (cDNA) synthesized from RNA by reverse transcriptase.
Genetic Selection - ANSW-searching for a DNA sequence that restores the wild-type phenotype to a
mutant organism
Molecular Hybridization - ANSW-Molecular hybridization is based on the hybridization of similar DNA
sequences
Cloning Vector - ANSW-- a small piece of DNA that can be stably maintained in an organism, and into
which a foreign DNA fragment can be inserted for cloning purposes.
- used to obtain numerous copies of inserted DNA segment
- can be plasmids, cosmids, phages, BACs, YACs, or MACs
- comprise of an OriC, unique restriction site, and selectable marker
Expression Vector - ANSW-- a plasmid that is used to introduce a specific, foreign gene into a target cell
and carry out expression of the gene by commandeering the molecular machinery of the target cell
- used to obtain gene product of DNA insert (either RNA or protein)
- this is a plasmid vector
- comprises of enhancers, termination codon, transcription initiation sequence, and translation initiation
sequence along with all of the components of a typical cloning vector
Southern blotting - ANSW-Molecule Detected: DNA
Gel Electroph: Agarose
Gel Pretreatment: Depurination, denaturation, and neutralization
Blotting method: capillary transfer
Probes: DNA radioactive or nonradioactive
Northern blotting - ANSW-Molecule Detected: mRNA
Gel Electrop: Formaldehyde agarose
Gel Pretreatment: None
Blotting Method: Capillary transfer
Probes: cDNA, cRNA radioactive or nonradioactive
Western blotting - ANSW-Molecule Detected: Protein
Gel electroph: polyacrylamide
