Polymerase chain reaction (PCR), DNA sequencing Flashcards
What are some uses of PCR?
- Sequencing (some approaches)
- DNA cloning (isolating a particular gene)
- Detection of pathogens
- Gene editing
PCR generally depends upon knowing the nucleotide
sequences at the ______ of the region to be amplified.
ends
What is the composition of a single reaction tube in PCR? (4)
-DNA template (can be a complex mixture, like total DNA from a cell)
-DNA polymerase that is stable at high temperature
-Primers complementary to each end of the region to be amplified (oligonucleotides or oligos)
-dNTPs
What are the three steps involved in a PCR cycle?
-denaturation: temperature is increased to separate DNA strands
-annealing of primers: temperature is decreased to allow primers to base pair to complimentary DNA template
-extension by DNA polymerase: Polymerase extends primer to form nascent DNA strand
What is Taq polymerase?
The most frequently used DNA polymerase
Why is Taq polymerase best for amplifying short fragments?
is thermophilic and cheap but does not have proofreading exonuclease activity
Why do we have to repeat the 3 steps cycle multiple times?
Exponential amplification allows for:
-very sensitive detection of a DNA sequence in the sample
-enables purification of substantial amounts of a specific DNA fragment for further use
Why do we use gel electrophoresis in PCR?
to separate DNA fragments produced by PCR based on their
size.
What is the Dideoxy Chain-Termination Method of
DNA Sequencing? (also known as Classical Sanger
Sequencing)
Sanger sequencing is designed for determining the sequence of nucleotide bases in a piece of DNA
What are the contents of ALL 4 tubes used in Sanger sequencing? (4)
DNA Polymerase
Oligonucleotide primer
DNA template
dNTPs (100 mM)
What is added in one of each 4 tubes ?
chain terminators:
ddATP (1 mM)
ddGTP (1 mM)
ddTTP (1 mM)
ddCTP (1 mM)
What is a chain terminator?
The ddNTPs don’t have the 3’OH and instead have a 3’H which allows to stop the sequencing of DNA. (DNApol will fall off)
What are the limitations of Sanger sequencing? (2)
- Polymerase only runs for 300-500 nucleotides, and gels can only clearly resolve this much, so many separate individual sequencing reactions must be run to sequence a large region.
- The rate of sequence production is limited by the total number of reactions that can be performed at one time.
What is Next-generation sequencing (NGS)?
- NGS represented a technological breakthrough. Methods were developed to allow a single sequencing instrument to carry out millions of sequencing reactions simultaneously.
How does one type of NGS technology work? (4)
-One type of NGS technology involves ligating the same linkers to a mixture of DNA fragments.
-The DNA is then denatured and annealed to complementary primers anchored to a solid support.
-PCR is then conducted, amplifying the DNA fragments in a fixed spatial arrangement.
-Next, the double stranded DNA is cut and only one strand is sequenced, with fluorescently labeled dNTPs (different colour for each base). Imaging and removal of fluorophore takes place after each cycle.
