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?
