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41 Cards in this Set
- Front
- Back
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What is the template for a PCR reaction?
How many primers do you need for typical PCR? Where do you get them? |
genomic DNA
TWO primers, about 20-30 base pairs long- must be perfectly complementary to template strands. They just order primers from a company. |
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How many primers do you need for PCR?
How many primers do you need for PCR associated with Sanger sequencing? |
You need TWO primers for normal PCR. ONE primer ("oligo") for Sanger sequencing.
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What do you need to run a PCR?
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1. DNA polymerase (normally Taq polymerase)
2. Genomic DNA 3. dNTP's 4. oligonucleotide primers |
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Name (describe with nomenclature) a mutation that switches T to A , two bases down from the start of the 3rd intron.
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g.IVS3+2T>A
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Name (describe with nomenclature) the deletion of an AAAA sequence at positions 1234, 1235, 1236, 1237 in mRNA.
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c.1234_1237delAAAA
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Describe (describe with nomenclature) a mutation that substitutes of an aspartate with an asparagine at position 212 in a polypeptide.
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Asp212Asn
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What is happening at 95 degrees during PCR?
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genomic DNA is being denatured
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What is happening at 55 degrees in PCR?
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primers are annealing to targets on a template
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Why don't the original strands of DNA reanneal instead of annealing with the manufactured primers in PCR?
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You flood the rxn with a very high concentration of primers - they will find their target site on the template strands much faster than the original complement strand will.
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Why do they use Taq DNA polymerase instead of human DNA pol in a PCR rxn?
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DNA must be denatured at a high temperature - need a Polymerase that won't degrade at high temp.
Also Taq DNA pol works optimally to make complement at 72 degrees. |
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What is the sequence of temperature changes in a PCR rxn - what happens at 72 degrees?
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95 degrees = denatures genomic DNA
55 degrees = primers anneal to targets 72 degrees = complement strand is made |
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What are 3 uses for PCR?
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1. amplify a suspected mutation for further study.
2. generate VNTR's for fingerprinting 3. use ASO for PCR primer so only product you're looking for will appear if primer matches it = ARMS |
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What is VNTR?
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Variable Number of Tandem Repeats
each person has a different number of repeating sequences. You can use this with PCR to ID someone's DNA from a crime scene. |
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What is ARMS?
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Amplification Refractory Mutation System.
Use ASO for PCR primer (ONE primer needed) so only the product you're looking for will appear if primer matches it. |
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What is Sanger sequencing?
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Run PCR using low concentration of fluorescently tagged ddNTP's. You will get varying lengths of complement strands depending on what ddNTP the primer picked up. The Sanger sequencing part is when you use the tags on the terminal nucleotides of each complement to determine the sequence of the strand based on length.
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Why are ddNTP's used in Sanger sequencing?
What is a ddNTP? |
ddNTP's don't have an -OH group on their 3' carbon. With no 3' carbon - another nucleotide will not attach to it to form DNA. It effectively stops transcription. This makes it possible to determine a sequence based on labeled ddNTP's and then length of complement they create.
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When using Sanger sequencing is it important to have excess ddNTP's or excess dNTP's?
Why? |
You need excess dNTP's. You won't make enough complement strands if you don't saturate the rxn with dNTP's. ddNTP's will not link together at all.
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Which assay uses probes at a single locus to see if a gene has been removed? How is this done?
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Single gene FISH.
You tag a gene with fluorescent probes - if you see the tag on one chromosome and not the other - you know that gene has been deleted. (Karyotyping) |
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What does DNA microarray test for?
Where are the samples put? |
DNA microarray detects changes in gene copy number and expression. Samples put on a microscope slide.
Can also be used to measure changes in RNA using cDNA. |
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Which high energy bond is formed during the first aminoacyl tRNA synthetase rxn?
What is the deltaG for that rxn? What pays for this entropically? |
The carboyxlphosphate bond between the terminal --O- on the AA and the 5' phosphate group of AMP.
deltaG= +12 Hydrolysis of two release pyrophosphate bonds. deltaG= -15 |
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What is the first step in the aminoacetylation of tRNA?
What is the purpose of this rxn? |
amino acid + ATP (aaRS)---> aminoacyl-AMP + PPi
This rxn creates a high-energy carboxylphosphate bone which in turn will activate the carboxyl C of the AA for attack by the 2' or 3' OH of the 3' end of tRNA. |
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What is the second step in the aminoacetylation of tRNA?
Is deltaG positive or negative for this rxn? |
aminoacyl-AMP + tRNA (aaRS)---> aminoacyl-tRNA + AMP
deltaG is negative for this rxn bc formation of the ester bond linking tRNA to the AA is much lower energy than the carboxylphosphate bond bt the AMP and the AA. |
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What kind of bond links an amino acid to an AMP? (rxn one)
What kind of bond links an aminoacyl-AMP to a tRNA? (rxn 2) |
A high-energy carboxylphosphate bond links molecules in rxn one.
A lower energy ester bond links molecules in rxn 2. |
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What is the start codon? What AA corresponds with this? Where does it attach to the ribosome during translation?
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AUG codes for Methionine =
methionl-tRNA-met(i) ***This is the only aminoacyl-tRNA that binds to the P site!!! All other aminoacyl-tRNA's bind to the A site. |
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Where does proof-reading occur in aminoacetylation of tRNA?
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if aminoacyl-AMP doesn't fit into the right site, AMP will be hydrolyzed off and the AA thrown out.
this can happen before or after it becomes aminoacyl-tRNA. |
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Where does initiation begin in translation in eukaryotes?
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Initiation begins at the 1st AUG downstream of the 5'-cap on mRNA.
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What enzyme catalyzes the peptidyltransferase rxn?
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THE LARGE SUBUNIT OF THE RIBOSOME. It acts like an enzyme= a ribozyme.
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Describe the peptidyltransferase rxn.
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This rxn is what removes AA's from tRNA and attaches them to each other.
The free amino group on the A-site tRNA attacks the ester bond bt the AA and the tRNA on the P-site. |
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Which is a higher energy bond, ester bond in aminoacyl-tRNA or a peptide bond in an amino acid?
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the ester bond is higher energy...this is significant because this make the peptidyltransferase rxn favorable.
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Name two translation factors.
Both of these are themselves regulated by what? |
EF-1alpha and EF-2.
Both are regulated by GTP. |
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EF-1alpha functions as another _______ step.
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EF-1alpha functions as another proof-reading step.
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How does EF-1alpha act as a proof-reading step?
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EF-1a binds to an aa-tRNA and goes with it to the A site where the GTP attached to it is hydrolyzed. Then EF-1a-GDP is released from the aa-tRNA. PEPTIDE BOND WON'T FROM until this happens. This rxn takes time- during this time incorrect aa-tRNA's will dissociate.
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What is EF-2's function?
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EF-2 is a GTP regulated protein that is necessary for translocation of the peptidyl-tRNA from the A site to the P site.
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How does Diphtheria kill you?
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Corynebacterium diphtheria releases a toxin that inactivated EF-2. This blocks protein synthesis (prevents translocation).
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What signals the hydrolysis of GTP on the EF-1a?
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The binding of the aatRNA to the A site.
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99% of human introns start with what sequence?
End with what? |
Start with GU and end in AG.
ex) GU.........................AG |
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What is the proper definition of exon?
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An exon is a segment of primary transcript which is NOT removed.
***It may include some material that is not translated into protein. |
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What first recognizes the 5' splice site?
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U1 snRNA first recognizes the 5' splice site.
Preferred adjacent nucleotides do help attract the U1 snRNA to the site. |
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How can alternative splicing change the mRNA without changing the N or C terminal domains?
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mutually exclusive exons
"cassette exons" |
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How can alternative splicing cause a change in the carboxy terminus?
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pre-mRNA can have multiple Poly(A) sites for different forms of expression of that gene.
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εἰς
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unto; into; to; in (+acc.)
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