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The Sanger Method is also known as what?
The Dideoxy-Method
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The Dideoxy Method uses dideoxynucleotides to do what?
To terminate synthesis of DNA at specific nucleotides.
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The Dideoxy Method uses dideoxynucletodies as what?
Chain Terminators
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Dideoxy-sequencing is based on DNA or RNA replication?
DNA
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For DNA replication, where is the incoming nucleotide added?
To the 3' OH (Hydroxyl group) on the growing primer strand
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What happens when a dideoxynucleotide has been added during DNA synthesis?
The synthesis of that strand is terminated and no more nucleotides can be added.
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What does the Dideoxy Method require?
- The DNA to be sequenced
- A synthetic DNA primer
- the 4 dNTPs
- a DNA polymerase
- a low concentration of ddNTPs
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How many base pairs can the 454 Sequencing Method generate per run?
100 Mb
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Whats the key event in Pyrosequencing Reactions?
The detection of the release pyrophosphate molecule (PP_i) as each dNTP is incorporated.
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The PP_i is a substrate for what?
for ATP-sulfurase
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The PP_i is a substrate for ATP-sulfurase and produces what?
ATP
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What happens to the ATP?
It is hydrolized to ADP in a light production reaction
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What does SMRT sequencing stand for?
Single-Molecule Real-Time
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Where are the flourescent labels locatd on the four different dNTPs?
gamma-phosphates
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What does that mean?
It means that the nucleotide is labeled as it is being held by the polymerase but not after it has been incorporated into the DNA
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What is the labeled nucleotide detected through?
Nanophotonic Zero-Mode Waveguides (ZMWs)
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What does PCR stand for?
Polymerase Chain Reaction
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PCR was developed in the 1980s by who?
Karry Mullis
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What reaction components are added at the start of PCR reaction? (Before the cycles.)
- Template
- Primers
- dNTPs
- DNA polymerase
- Buffer
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List the steps of each cycle in PCR (the basic method).
- 1) Heat the Target DNA strand.
- 2) Cool the Target DNA strand to ~55C.
- 3) Heat to 72C
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For PCR, what does heating the target DNA strand do?
It denatures the DNA to form single strands.
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What does cooling the target DNA to ~55C do?
Makes the added Primers anneal to the target DNA.
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What does heating the target DNA strand to 72C do?
It's the optimum for heat-stable DNA polymerase.
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In Cycle 2 of PCR, what happens?
The same steps are repeated, doubling the amount of the target sequence.
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With Cycle 3 and above, what do the amplified target sequences have?
They have a length defined by the primer sites, therefore products can be analyzed or isolated on a gel.
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So, what can PCR let you do? (3 things)
- 1) PCR can be used to detect a specific DNA sequence in a complex mixture.
- 2) PCR can be used to isolate a specific fragment of DNA.
- 3) It can be used to detect mRNAs, isolating cDNAs, and quantifying any nucleic acid.
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What does PCR need in order to detect mRNAs?
Use Reverse Transcriptase.
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What's an example of PCR being used?
Linkage Analysis using individual Sperm
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What do Restriction Endonucleases do?
Cut in the middle of DNA.
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What are the most common restriction enzymes?
"Type II" enzymes.
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Restriction Fragments with compatible ends can be what?
Ligated to form new recombinant DNAs
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What does DNA ligase do?
Forms phosphodiester bonds to covalently bond the fragments into one double stranded DNA
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What type of cut DNA is made with restriction enzyme SmaI?
Blunt Ends are made.
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What type of cut DNA is made with restriction enzyme BamiIII?
5' overhanging (sticky) ends
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What type of cut DNA is made with restriction enzyme PstI?
3' overhanging (sticky) ends
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We can predict how frequently an enzyme will cut DNA based on what?
Based on the size of its recognition sequence.
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Electrophoresis is a method to do what?
To separate molecules based on size, conformation, or charge density.
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For Electrophoresis, agrarose gel is used for smaller or larger fragments?
Larger
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For Electropheresis, acrylamide gel is used for smaller or larger fragments?
Smaller
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Nucleic Acid Hybridization is used for what?
To detect the DNA or RNA of a specific sequence
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Nucleic Acid Hybridization is used by which DNA Methods?
- Southern & Northern Blotting
- PCR and PT-PCR
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What does RFLPs stand for?
Restriction Fragment Length Polymorphisms
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RFLPs provide what?
Molecular markers that can be followed in crosses
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What results in a detectable polymorphism?
Any mutation that elimates or creates a restriction site or that adds or deletes a significant amount of DNA from a restriction fragment
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What is the difference between Northern & Southern Blotting?
- In Northern Blotting, RNA is separated by electrophersis, transferred to a membrane, the specific sequences are detected by hybridization.
- In Southern Blotting, it's DNA.
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True or False. Restriction Enzymes are required for RNA in Northern Blotting.
False
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What does Western Blotting detect?
Specific proteins in a complex mixture.
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What occurs in Western Blotting?
- Proteins are first electrophoresed in a polyacrylamide gel.
- Transferred to a memrbane.
- Then specific proteins are detected.
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How are the specific proteins detected in Western Blotting instead of using Hybridization?
Using antibodies.
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RT-PCR stands for what?
Reverse Transcriptase Polymerase Chain Reaction
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RT-PCR is an alternative for what?
Northern Blotting
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What does PT-PCR do?
Detects the presence of a specific RNA in a complex sample mixture.
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PT-PCR is essentially identical to conventional PCR except that the first step is to use the enzyme what?
Reverse Transcriptase
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What does Reverse Transcriptase do?
Copies the mRNA into a single stranded cDNA (copy DNA)
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Also like conventional PCR, RT-PCR is a Qualitative or Quantitative method?
Qualitative
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Is Northern Blotting qualitative or quantitative?
Quantitative
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Which is faster? PCR or Northern?
PCR
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