BIOL360 DNA Methods
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The Sanger Method is also known as what?
The Dideoxy Method uses dideoxynucleotides to do what?
To terminate synthesis of DNA at specific nucleotides.
The Dideoxy Method uses dideoxynucletodies as what?
Dideoxy-sequencing is based on DNA or RNA replication?
For DNA replication, where is the incoming nucleotide added?
To the 3' OH (Hydroxyl group) on the growing primer strand
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.
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
How many base pairs can the 454 Sequencing Method generate per run?
Whats the key event in Pyrosequencing Reactions?
The detection of the release pyrophosphate molecule (PP_i) as each dNTP is incorporated.
The PP_i is a substrate for what?
The PP_i is a substrate for ATP-sulfurase and produces what?
What happens to the ATP?
It is hydrolized to ADP in a light production reaction
What does SMRT sequencing stand for?
Where are the flourescent labels locatd on the four different dNTPs?
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
What is the labeled nucleotide detected through?
Nanophotonic Zero-Mode Waveguides (ZMWs)
What does PCR stand for?
Polymerase Chain Reaction
PCR was developed in the 1980s by who?
What reaction components are added at the start of PCR reaction? (Before the cycles.)
- DNA polymerase
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
For PCR, what does heating the target DNA strand do?
It denatures the DNA to form single strands.
What does cooling the target DNA to ~55C do?
Makes the added Primers anneal to the target DNA.
What does heating the target DNA strand to 72C do?
It's the optimum for heat-stable DNA polymerase.
In Cycle 2 of PCR, what happens?
The same steps are repeated, doubling the amount of the target sequence.
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.
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.
What does PCR need in order to detect mRNAs?
Use Reverse Transcriptase.
What's an example of PCR being used?
Linkage Analysis using individual Sperm
What do Restriction Endonucleases do?
Cut in the middle of DNA.
What are the most common restriction enzymes?
"Type II" enzymes.
Restriction Fragments with compatible ends can be what?
Ligated to form new recombinant DNAs
What does DNA ligase do?
Forms phosphodiester bonds to covalently bond the fragments into one double stranded DNA
What type of cut DNA is made with restriction enzyme SmaI?
Blunt Ends are made.
What type of cut DNA is made with restriction enzyme BamiIII?
5' overhanging (sticky) ends
What type of cut DNA is made with restriction enzyme PstI?
3' overhanging (sticky) ends
We can predict how frequently an enzyme will cut DNA based on what?
Based on the size of its recognition sequence.
Electrophoresis is a method to do what?
To separate molecules based on size, conformation, or charge density.
For Electrophoresis, agrarose gel is used for smaller or larger fragments?
For Electropheresis, acrylamide gel is used for smaller or larger fragments?
Nucleic Acid Hybridization is used for what?
To detect the DNA or RNA of a specific sequence
Nucleic Acid Hybridization is used by which DNA Methods?
- Southern & Northern Blotting
- PCR and PT-PCR
What does RFLPs stand for?
Restriction Fragment Length Polymorphisms
RFLPs provide what?
Molecular markers that can be followed in crosses
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
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.
True or False. Restriction Enzymes are required for RNA in Northern Blotting.
What does Western Blotting detect?
Specific proteins in a complex mixture.
What occurs in Western Blotting?
- Proteins are first electrophoresed in a polyacrylamide gel.
- Transferred to a memrbane.
- Then specific proteins are detected.
How are the specific proteins detected in Western Blotting instead of using Hybridization?
RT-PCR stands for what?
Reverse Transcriptase Polymerase Chain Reaction
RT-PCR is an alternative for what?
What does PT-PCR do?
Detects the presence of a specific RNA in a complex sample mixture.
PT-PCR is essentially identical to conventional PCR except that the first step is to use the enzyme what?
What does Reverse Transcriptase do?
Copies the mRNA into a single stranded cDNA (copy DNA)
Also like conventional PCR, RT-PCR is a Qualitative or Quantitative method?
Is Northern Blotting qualitative or quantitative?
Which is faster? PCR or Northern?
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