Lab 2: PCR Flashcards
The polymerase chain reaction (PCR) was developed in 1983 by
Kary Mullis
is a method for producing an extremely large numbers of copies of a specific DNA sequence from a small amount starting material
PCR
Before PCR, DNA amplification had to be done through a difficult and time-consuming process called
DNA cloning
producing an extremely large numbers of copies of a specific DNA sequence from a small amount starting material
amplification
In DNA cloning, the DNA segment to be amplified is first moved into a
shuttling vector
would then need to be cut back out of the vector and re-purified.
amplified fragment
PCR limitations
potential for indiscriminate amplification of contaminant DNA.
it requires one to know the nucleotide sequence of the region to be amplified.
Without DNA sequence information, primers can not
be designed
The components of a typical PCR reaction are as follows:
1) A source of DNA
2) Two synthetic primers that are specific for the region of DNA to be amplified.
3) All four deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, dGTP).
4) An appropriate reaction buffer
5) A thermostable DNA polymerase enzyme.
Primers are typically between how many nucleotides long
18-30
is designed to be complimentary to the left flanking end of the region to be amplified
forward primer
is designed to be complimentary to the right flanking end
reverse primer
All four deoxyribonucleotide triphosphates
dATP, dTTP, dCTP, dGTP
You can’t make new DNA without new
nucleotides
temperature that normally denature most types of proteins
(95°C)
example of A thermostable DNA polymerase enzyme
Taq polymerase
After mixing all of the above components together into a small plastic tube, the reaction mix is placed into a machine called a
thermal cycler
what does the thermal cycler do
functions to cycle the mix through the three main steps of PCR
three main steps of PCR
1) Denaturation of the template DNA strand
2) Annealing of the primers to the template strands
3) Extension of primers by Taq polymerase
The two strands that make up DNA must be
what in order for amplification to take place
separated (denatured)
The quickest/cheapest way to achieve total DNA denaturation
heating the strands to 95°C
for 1-1.5 minutes.
Heating the samples hotter/longer than that reduces
the activity of the
Taq polymerase
heating it shorter/quicker reduces the
overall efficiency of the amplification (due to poor denaturation)
lowering the temperature of the reaction tube to an appropriate level to allow the two primers to
anneal to the complimentary regions on the template strands
proper temp fr annealing
usually 55-65°C for 1 min
If the annealing temperature is set too low
the primers tend to anneal non-specifically at random locations on the template
Setting the temperature too high
may prevent the primers from sticking at all
the primers direct the what to where to begin creating new DNA
Taq polymerase
With primers bound to the appropriate sites on the template, Taq will bind to the
3’ end of each primer and begin adding on new nucleotides (if it reads a G, it will add a C across from it
This extension of the primers in the what direction
5’ to 3’ direction
extention will continue until
the polymerase reaches the end of the template strand
or
it simply falls off after going a long distance.
amplification is
exponential
