U4 LAB: POLYMERASE CHAIN REACTION Flashcards
Two big issues in Identification of a Specific Sequence
- Specificity
- Amplification
E. coli
4.6 million bp
HIV
9.7 thousand bp
Ability to detect the target DNA strand to a group of various DNA strands
Specificity
Avogadro’s number
6.02^23
PCR was developed by?
Kary Mullis in mid 1980s
Amplification is done?
exponentially
every product will be used for the next cycle
A relatively simple technique developed in 1985 to amplify sequence-specific DNA fragments in vitro
Polymerase Chain Reaction (PCR)
This is an enzymatic process in which a target DNA sequence is copied by?
DNA polymerase
PCR amplification is achieved by using?
short, single stranded, oligonucleotide primers
Serve as primers for DNA polymerase
oligonucleotides
Serves as template
Denatured strands of the large DNA fragment
The shorter the primer, the ________ the process
faster
T/F: PCR can amplify a specific subsequence out of a much larger DNA.
True
T/F: Each PCR cycle theoretically doubles the amount of DNA.
True
Components for PCR Amplification
- PCR
- Template DNA
- Target specific forward and reverse oligonucleotide primers
- PCR buffer with MgCl2
- Each of the 4 dNTPs
- Taq polymerase
PCR buffer is mixed with?
MgCl2 (cofactor for DNA polymerase)
under dNTPs
dATP, dCTP, dGTP, dTTP
This refers to the thermostable DNA polymerase.
Taq polymerase
Temp for Primer Annealing
50 to 65
30 is seldom used
Temp for Denaturation
94-95
Temp for Primer Extension
72
As temperature is raised, the DNA strands?
separate or denature
The temperature is then lowered to?
allow primers to attach or anneal
The temperature is raised after annealing to provide optimum environment for a special heat resistant type of DNA polymerase called?
Taq polymerase
Taq polymerase stands for?
Thermus aquaticus
After five cycles, _______ copes have been produced.
32
Taq polymerase binds to?
double-stranded substrate
This is a perfected version in which there is no more excess after cycles, only contains the target DNA sequence.
amplicons
This effect happens in the absence of a primer.
Plateau effect
Affinity to detect DNA target sequence if you have a large amount of DNA sample
Specificity
Ability to detect target sequence if you have a small amount of DNA sample
Sensitivity
Low Mg2+
increase specificity, decreased yield
High Mg2+
increase sensitvity, decrease specificity
Too much DNA polymerase?
decreases specificity
Too little DNA polymerase?
insufficient product
Pfu polymerase stands for?
Pyrococcus furiosus
Antibody binds and inactivates Taq polymerase at?
room temperature
Polymerase is activated when?
heat denatures and releases antibody
This technique prevents formation of primer-dimers and other non-specific products
Hot Start Techniques
This is when 2 primers anneal each other instead to the intended DNA sequence.
Primer-dimers
Denaturation temp
High can?
increase sensitvity
Denaturation temp
Taq pol activity can?
decrease above 93C
Primer annealing temp
Increase in temp?
increases specificity
Primer annealing temp
Decrease in temp?
increases sensitivity
Duration time of primer extension
Longer times increase?
sensitivity
Cycle number
_____ number of cycles if rxn has _____ initial target molecules
Increase, <103
PCR inhibitors interact?
directly with DNA or interfere with DNA polymerases
PCR inhibitors are endogenous to sample or introduced during?
sample processing or DNA purification
Common inhibitors
- Hemoglobin
- Urea
- Heparin
