Midterm 4 Flashcards
Define pyrosequencing.
A parallel strategy enabling hundreds of thousands of short sequences to be sequence at a time.
Define chain termination DNA sequencing.
Based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another by gel electrophoresis.
What is the starting material for chain termination DNA sequencing.
preparation of identical single-stranded DNA molecules.
How does dideoxynucleotide work?
blocks further elongation because it lacks the 3′-hydroxyl group needed to form a connection with the next nucleotide.
Why does strand synthesis not end close to the primer in chain termination DNA sequencing?
Because the normal deoxynucleotides are also present, in larger amounts than the dideoxynucleotides
What is the result of chain termination DNA sequencing?
a set of new molecules, all of different lengths, and each ending in a dideoxynucleotide whose identity indicates the nucleotide—A, C, G, or T
How can the DNA sequence be determined?
identify the dideoxynucleotide at the end of each chain-terminated molecule by running the strands through a gel.
The mixture is loaded into a well of a gel, and electrophoresis is carried out to separate the molecules according to their lengths.
After separation, the molecules are run past a fluorescent detector capable of discriminating the labels attached to the dideoxynucleotides The detector determines if each molecule ends in an A, C, G, or T.
Why can you not use all polymerases for chain termination DNA sequencing.
many DNA polymerases have a mixed enzymatic activity, being able to degrade as well as synthesize DNA (p. 48). Degradation can occur in either the 5′→3′ or 3′→5′ direction , and both activities are detrimental to accurate chain termination sequencing.
What happens to degradation in the 5’?
What happens to degradation in the 3’?
The 5′→3′ exonuclease activity enables the polymerase to remove nucleotides from the 5′ ends of the newly-synthesized strands, changing the lengths of these strands so that they no longer run through the polyacrylamide gel in the appropriate order.
The 3′→5′ activity could have the same effect, but more importantly will remove a dideoxynucleotide that has just been added at the 3′ end, preventing chain termination from occurring.
What is the first step of a chain termination sequencing experiment?
A primer is annealed onto the template DNA.
What is the main function of the primer?
to provide the short double-stranded region that is needed in order for the DNA polymerase to initiate DNA synthesis.
also plays a second critical role in determining the region of the template molecule that will be sequenced.
Up to how many sequences can be obtained through a single run of chain termination sequencing experiment?
96 sequences
What primers are used if a plasmid vector was used to clone DNA?
The forward and reverse primers used in PCR. For example, we use T7 FP and SP6 RP.
True or False: The DNA polymerase used in subcloning has proofreading activity.
True
Define 3’-5’ exonuclease proofreading activity
Proofreading activity outside the DNA fragment
Define 3’-5’ endonuclease proofreading activity.
Proofreading activity inside the DNA fragment.
Define translesion syntehsis.
Direct replication of DNA damage
Explain DNA polymerase proofreading acticity
a spell-checking activity enabling DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus (3’) before further primer extension and prevents translesion synthesis.
What does sanger sequecning allow for?
Reading of DNA
What is the principle of sanger sequencing?
DNA synthesis/ replication
What did sanger wanted to do with DNA synthesis?
Wanted to carry out DNA synthesis in vitro (outside of cell, usually in test tube) rather than in vivo (in a cell).
What is needed in invivo DNA synthesis
Template DNA
Primer: need a primer to bind to each end (F and R)
Enzyme: DNA polymerase (Taq DNA polymerase in the lab)
dNTP: dATP, dGTP, dCTP, dTTP (the d stands for deoxy)
What is needed for sanger sequencing?
Template DNA
Primer: need a primer to bind to each end (F and R)
Enzyme: DNA polymerase (Taq DNA polymerase in the lab)
dNTP: dATP, dGTP, dCTP, dTTP (the d stands for deoxy)
ddNTP for Sanger sequencing
Draw the strcuture of dNTP and ddNTP. What is different?
What is used in DNA to make a phosphodiester bond?
In DNA the hydroxyl group of the dNTP is used to make a phosphodiester bond
What did sanger do to dNTP.
Removed the OH from the 3’ resulting in ddNTP
What happens when a ddNTP is incorporated in Sanger Sequencing?
Aphosphodiester bond cannot form and the strand stops since an additional nucleotide cannot be added to continue the strand.
What are the differences between what the dNTP and ddNTP allows? How do they differ?
dNTP allows for elongation while ddNTP allows for stopping.
dNTP has a OH in the 3rd position while ddNTP has a H.
What are the ends in fragments of Sanger sequencing like?
The 3’ ends different for each by a nucleotide.
how is the nucleotide at the end of the fragments in sanger sequencing determined?
By the ddNTP that was incorporated.
What do all the new strands from sanger sequencing have in common and different? How do we know the difference?
A common 5’
Different 3’ by one nucleotide
To know the difference, need to run a gel to know the nucleotide at the end.
What is the orientation of primers?
5’ to 3’
What direction are primers always made from the template strand?
3’ to 5’
How do you make a primer in a paragraph type sequence of nucleotides?
the reverse primer is the only one the primer is made from and the forward primer is a copy from the start codon.
What is in each sanger sequencing tube?
Template, primer, enzyme, dNTP, and ddNTP.
How many primers are used in sanger sequencing?
1, either reverse or forward.
When the tubes from sanger sequecing are loaded in a gel electrophoresis, what happens?
the shortest sequence will go first (to the bottom) then to the top (largest)
How is the sequence determined from a gel in sanger sequencing?
Read the sequence from bottom to top (5’ to 3’)
Then the complementary is the sequence
How many tubes are prepared in modern day sanger sequencing? What are in the tubes?
1 reaction
Template
Primer (F or R)
Taq DNA polymerase
dNTPs
Fluorescently labeled ddNTP
How many flourescent colors are emitted in sanger sequencing.? What do they correspond to?
4 colors corresponding to each nucleotide.
What is the molecular weight of an amino acid?
0.11 mol/g
What are the two parts of Consensus sequences?
Cofactor binding site and active site.
How many primers are needed a sequence from one side? What if you want to read a sequence from both sides?
If you just want to read a sequence, you need 1 primer (reverse or forward)
If you want to read a sequence from both sides need forward and reverse
Name the experiment and the result of Isolation in cloning.
Experiment - Polymerase Chain Reaction.
Deanture, Annealing, Extension
Result: Extracted the ORF of the CR gene.
How is a PCR verfied?
gel electrophoresis and measure concentration by getting OD using a nandrop spectrophotometer
What is in the master mix?
DNA polymerase, FP, RP, dNTPs, green color
Name the experiment and the result of Insertion in cloning.
Experiment - Ligation
Insert, vector, Dna ligase (T4 ligase), buffer.
Result: A recombinant DNA molecule with an insert (the extracted ORF of the CR gene). The insert is ligated to the PGEMT - Easy Vector.
What is done prior to ligation?
Purification
What are the buffers used in purification and their role?
PB: bind to column
PE: Contains ethanol to remove contaminants
EB: Unbind the DNA from the column and precipitate it.
What are the components of ligation?
Insert, vector, Dna ligase (T4 ligase), buffer.
Name the experiment and the result of amplification in cloning.
Experiment: Transformation
Result: LB agar plate + host cells with recombinant molecule + Xgal + IPTG
What happens in transformation?
Incubation in host cells (competent cells usually from e.coli, JM109)
Then plating to grow colonies
Name the experiment and the result of selection in cloning.
Experiment: Done through blue white screening.
Result: White colonies with recombinant DNA molecule
What is selected in blue white screening and why? Why is the colony that color?
Select white colonies due to them having the recombinant DNA due to insertional inactivation
Grow colonies
Name the experiment and the result of confirmation in cloning.
Experiment: Sanger sequencing
Result: Sequence of DNA
What is done prior to sequencing?
plasmid isolation and determine concentration through nanodrop
Why is confirmation done?
To verify presence of insert and if any mutations occurred
What is done after the sequence is obtained?
A blast to verify the insert is our gene of interest by comparing the sequence to the sequence in the ncbi database, to observe the orientation of the insert, and to observe whether there are any mutations.
What cloning did we use?
TA cloning
Name in detail the steps of cloning. Not just the 5
Isolation
PCR - Denaturing, Annealing, Extension
Gel electrophoresis
Nanodrop
Insertion
Purification
Nanodrop
Ligation
Amplification
Transformation: Incubation and plating
Selection
Blue white screening
Plasmid isolation
Nanodrop
Confirmation
Sanger sequencing
BLAST
Why did we use a cloning vector for the cloning step of expression? What is our ultimate goal
We were interested in making copies of the insert. Our ultimate goal is expression of the CR gene.
