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