[2S] UNIT 3 Polymerase Chain Reaction Flashcards
2 issues in identifying and detecting a specific sequence in a genome
Specificity & Amplification
The human genome is ___ billion base pairs
3.4 billion
T/F: PCR solves the issues of specificity and amplification.
T
- Developed by Kary Mullis in mid-1980’s
- A “copy machine” for DNA
- Revolutionized molecular biology
Polymerase Chain Reaction (PCR)
He was granted the nobel prize in chemistry in 1993 for Polymerase Chain PCR
Kary Mullis
Amplification of DNA
Denature → Anneal → Extend (repeat)
is a relatively simple technique developed in 1985 to amplify sequence-specific DNA fragments in vitro
PCR
one of the most useful techniques in laboratories today due to its speed and sensitivity.
PCR
PCR can be:
- performed in _ hr
- requires as little as _ DNA molecule
1 hr
1 DNA molecule
useful in basic research and commercial applications, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics
PCR
An enzymatic process in which a target DNA sequence is copied by DNA polymerase
PCR
T/F: Ideally 30-40 cycles are done in PCR
T
PCR
_______ ____ __________ increases exponentially at each cycle, as amplification products from each cycle become the template for the next round of amplification
Target DNA concentration
1,2,3,4,5,6,7,….
Linear Amplification (from the word itself)
1,2,4,8,16,32,64,128,…
PCR Amplification
is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence.
PCR
PCR amplification is achieved by using
oligonucleotide primers
These are typically short, single stranded oligonucleotides which are complementary
to the outer regions of known sequence
oligonucleotide primers
serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template.
oligonucleotides
T/F: Oligonucleotide Primers
This results in the synthesis of new DNA strands which are complementary to the
parent template strands.
T
can be used to target a specific DNA subsequence in a much larger DNA sequence (e.g., a single 1000bp gene from the human genome, which is 3 × 10^9 bp).
PCR
T/F: Oligonucleotide Primers
These new strands have defined 3’ ends (the 3’ ends of the oligonucleotide primers), whereas the 5’ ends are potentially ambiguous in length.
F; These new strands have defined 5’ ends (the 5’ ends of the oligonucleotide primers),
whereas the 3’ ends are potentially ambiguous in length.
allows exponential amplification of a DNA sequence
PCR
T/F: Each PCR cycle theoretically doubles the amount of DNA.
T
5 Components of PCR amplification
- Template DNA
- Target specific forward and reverse oligonucleotide primers
- PCR buffer (with MgCl2)
- Each of the four dNTP’s
- Thermostable DNA Polymerase
T/F: During PCR, an existing DNA molecule is used as a template to synthesize a new DNA strand.
T
T/F: Only one repeated rounds of DNA synthesis, large quantities of DNA are produced.
F; Through repeated rounds of DNA synthesis, large quantities of DNA are produced.
cofactor for DNA polymerase
Mg^2+
Denaturation temperature
94C, 30 secs to 1 min
Annealing temp
30-60C sa pic tas 50-65C sa ppt
This cycle is repeated and the DNA is copied exponentially
PCR: Thermal Cycling
Extension temp by DNA polymerase
72C
T/F: Anneal Primers
The temperature is then lowered to aloe primers – short, ssDNA molecules to
attach or anneal to the strands of DNA.
T
T/F: Extension of Primers
The temperature is raised again to provide the optimum environment for a special heat-resistant type of DNA polymerase
called Thermus aquaticus or Taq polymerase.
T
The temperature is raised again to provide the optimum environment for a special heat-resistant type of DNA polymerase
called?
Thermus aquaticus or Taq polymerase
After 5 cycles, __ copies of target DNA sequences have been produced. About 30 cycles are used to produce enough copies for further use.
22
,
To anneal primers to the template
Cool (60C)
Activate the Taq polymerase which
extends primers and replicates DNA
Warm 72C
↓Mg2 _ specificity
↑ specificity
stabilizes primer annealing, can increase
sensitivity, can decrease primer specificity
↑ Mg2
↑ Mg2 ↑ sensitivity
An antibody binds and inactivates Taq Polymerase at room temperature
Hot Start Techniques
Polymerase activated when heat denatures and releases antibody
Hot Start Techniques
Prevents formation of primer-dimers and other non-specific products
Hot Start Techniques
T/F: In denaturation temperature, Taq Pol activity decreases above 93C
T
longer duration time of primer extension _ sensitivity
↑ sensitivity
↑ Primer annealing temp _ specificity
↑
T/F: Only the complementary strand is left
T
efficiency of enzyme reaction, initial
number of DNA target molecules
Sensitivity
Inhibit amplification of nucleic acids by PCR
PCR Inhibitors
Interact directly with DNA or interfere with DNA polymerases
PCR Inhibitors
Endogenous to sample (blood, tissue, food) or introduced during sample processing or DNA purification
PCR Inhibitors
T/F: Detecting Inhibitors
Complete reaction failure (false negative) or reduced sensitivity
T
T/F: Detecting Inhibitors
Larger targets preferentially amplified
F; smaller
T/F: Detecting Inhibitors
Internal positive controls (IPC)
o Same reaction vessel versus separate vessel
T
T/F: Detecting Inhibitors
Internal positive controls (IPC)
o Monitor non-specific inhibition of nucleic acid amplification
T
T/F: Detecting Inhibitors
Internal positive controls (IPC)
o Exogenous/spiked sample or internal second target (housekeeping gene)
T
T/F: Detecting Inhibitors
Internal positive controls (IPC)
o Provide confidence in positive results
obtained in target-specific assays
F; negative results
