Sept 27 Polymerase Chain Reaction (PCR) DNA sequencing Flashcards
what does PCR do?
amplifies a specific DNA sequence, which can be part of a complex mixture
what are some uses of PCR?
sequencing (some approaches)
DNA cloning (isolating a particular gene)
detection of pathogen (such as SARS-CoV-2)
gene editing
what does the reaction tube contain?
DNA template (can be a complex mixture)
DNA polymerase that is stable at high temperature
primers complimentary to each end of the region to be amplified (oligonucleotides or oligos)
dNTPs
what are the three steps of the cycle that is repeated in PCR?
denaturation of DNA by heat, annealing of primers, and extension by DNA polymerase
which DNA polymerase is used in PCR?
Taq polymerase, originating from the thermophilic bacterium Thermus aquaticus
what is the limitation of Taq polymerase?
cheap but does not have proofreading exonuclease activity so it is best for amplifying short fragments
other enzymes with better fidelity can be used
what does exponential amplification allow for?
allows for very sensitive detection of a DNA sequence in the sample and purification of substantial amounts of a specific DNA fragment for further use
how are then DNA fragments produced by PCR further analysed?
can be analysed by gel electrophoresis
DNA restriction fragments are placed in the well of an agarose or polyacrylamide gel
an electric field is applied
molecules move through pores in the gel at a rate that is inversely proportional to their chain length (longer chain=slower)
each signal corresponds to a DNA band
what is the setup of the dideoxy chain termination method of DNA sequencing?
4 different tubes
each tube contains:
DNA polymerase
oligonucleotide primer
DNA template
dNTPs
in addition, each tube as one of the following chain terminators:
ddATP
ddGTP
ddCTP
ddTTP
what is the difference between a dNTP and a ddNTP and how does the latter act as a chain terminator?
ddNTPs do not have an OH group on the 3’ carbon (only a hydrogen)
that means that no further nucleotides can be added there –> chain terminator
what are the limitations of sanger sequencing?
polymerase only runs for 300-500 nucleotides, and gels can only clearly resolve this much
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
how does next generation sequencing (NGS) work?
NGS allows a single sequencing instrument to carry out millions of sequencing reactions simultaneously
the same linkers are ligated to a mixture of DNA fragments
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 labelled dNTPs (different colour for each base)
imaging and removal of fluorophore takes place after each cycle
what do even newer sequencing techniques allow for?
the PCR step can be skipped
a single molecule can be sequenced
how does dideoxy chain termination method of DNA sequencing work?
since the ggNTP is in low concentration, sometimes the normal nucleotide will bind, based on complementary base pairing
however, there will be a stop at every nucleotide of that certain type
the products of all 4 tubes are run through gel and arranged by size
following from band to band (next is one nucleotide longer than the previous), the template strand can be sequenced based on complementary base pairing
how does the nanopore technology work?
DNA (single strand) moves through motor protein
an electric current passes through the pore and is modulated as DNA passes through
the electric current is measured, and it varies depending on the base that moves through the protein
