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What is molecular cloning?
Isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated.
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What are the three steps of gene cloning?
- 1) Isolation and fragmentation of source DNA
- 2) Inserting DNA fragment into cloning vector
- 3) Introduction of cloned DNA into host organism
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What is the first step of gene cloning?
Isolation and fragmentation of source DNA
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What is the second step of gene cloning?
Inserting DNA fragment into cloning vector
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Most vectors are derived from ____
Plasmids or viruses
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DNA is generally inserted ____
In vitro
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What is the function of DNA lygase?
Enzyme that joins two DNA molecules together
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What is the third step of gene cloning?
Introduction of cloned DNA into organism
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____ is often used to get recombinant DNA into host
Transformation
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What are plasmids?
Natural vectors that have useful properties as cloning vectors
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What are the properties of plasmids?
- -Small size; easy to isolate DNA
- -Independent of origin of replication
- -Multiple copy number; get multiple copies of cloned gene per cell
- -Presence of selectable markers
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Plasmids are ____ of origin of replication
Independent
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What is Blue/White screening?
- Blue colonies do not have foreign DNA inserted into vector
- White colonies have foreign DNA inserted
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When plasmids are used as cloning vectors, _____ colonies do not have foreign DNA inserted into vector while ____ colonies had foreign DNA inserted into vector
Blue ; white
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What is insertional activation?
- lacZ gene is inactivated by insertion of foreign DNA
- -Inactivated lacZ cannot process Xgal; blue color does not develop
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When inactivated lacZ cannot process Xgal, _____ does not develop
Blue color
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What is the chemical process of insertional activation?
B-galactosidase --> x-gal substrate --> produces blue color
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What is an example of a common cloning vector?
pUC19
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What is pUC19 and what does it contain?
- 1) Common cloning vector
- 2) Contains ampicillin resistance and lacZ gene
- 3) Contains polylinker (multiple cloning site) within lacZ gene
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What is a polylinker and where is it found?
Multiple cloning site within lacZ gene ; found in pUC19 cloning vector
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What is sequencing?
Determining the precise order of nucleotides in a DNA or RNA molecule
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_____ is determining the precise order of nucleotides in a DNA or RNA molecule
Sequencing
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What is the Sanger dideoxy method and what is it used for?
- -Uses dideoxy analogs of dNTPs used in conjunction with dNTPs
- -Analog prevents further extension of DNA chain
- -Used for sequencing unknown DNA
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How do you make the DNA primer for unknown sequences?
Use random hexomers (random primers)
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What are hexomers used for?
Random DNA primers used for sequencing, using the Sanger method
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Why is the 454 sequencing system more advantageous?
It generates data 100X faster than the Sanger method
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The 454 sequencing system relies on two major advances. What are they?
- -Massively parallel liquid handling
- -Pyrosequencing
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In the 454 sequencing system, _____ is released each time a base is added to DNA strand
Light
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In the 454 sequencing system, light is released when ____
A base is added to the DNA strand
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In pyrosequencing, name the 4 enzymes involved in the reaction
- 1) DNA Polymerase
- 2) ATP sulfurylase
- 3) Luciferase
- 4) Apyrase
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In pyrosequencing, which enzyme converts PPi to ATPi?
ATP sulfurylase
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In pyrosequencing, which enzyme converts ATP to light?
Luciferase
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In pyrosequencing, which enzyme removes the nucleotides?
Apyrase
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In pyroseqencing, what is the function of ATP sulfurylase?
Converts PPi to ATPi
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In pyrosequencing, what is the function of Luciferase?
Converts ATP to light
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In pyrosequencing, what is the function of Apyrase?
Removes the nucleotides
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What is shotgun sequencing?
Entire genome is cloned and resultant clones are sequenced
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The process in which the entire genome is cloned and resultant clones are sequenced is called ____
Shotgun sequencing
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What is the difference between Closed and Draft genomes?
Closed genome relies on manpower while draft genome does not
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What does PCR stand for?
Polymerase Chain Reaction
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What is the Polymerase Chain Reaction (PCR)?
Method that produces multiple copies of DNA in vitro
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Where does the PCR process happen?
In vitro
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The method that produces multiple copies of DNA in vitro is called _____
Polymerase Chain Reaction (PCR)
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What enzyme does PCR use?
DNA Polymerase
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What is the name of the enzyme used for PCR and why is it used?
Taq Polymerase ; used because it is heat tolerant/thermostable
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In what instrument is PCR performed?
Thermocycler
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What are the three steps of PCR and at what temperatures do they occur?
- 1) Denaturation ; 94C
- 2) Annealing ; 50-60C
- 3) Extension ; 72C
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What is annotation?
Converting raw sequence data into a list of genes present in the genome
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The process of converting raw sequence data into a list of genes present in the genome is called _____
Annotation
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What is Functional ORF?
An open reading frame that encodes a protein
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An open reading fram that encodes a protein is called ____
Functional ORF
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In Functional ORF, computer algorithms search for what?
Start/stop codons, Shine-Dalgarno sequences, -35 and -10 regions
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What is the thermostable DNA Polymerase?
Taq polymerase
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Taq polymerase is stable at what temperature?
90C
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What is the difference between Taq polymerase and Pfu polymerase?
Pfu is more thermostable than Taq and Pfu has proofreading activity while Taq does not
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____ polymerase has proofreading activity while ____ polymerase does not
Pfu ; Taq
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During each round of PCR, the amount of product ____
Doubles
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Why is PCR an important technique?
- 1) Exponential increase in DNA
- 2) Only few molecules of target DNA are needed
- 3) PCR is valuable for cloning and sequencing purposes
- 4) PCR has been used to amplify DNA from mummified remains, fossilized plants and animals
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PCR has been used to amplify DNA from _____, _____ and ______
Mummified remains ; Fossilized plants ; Animals
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