[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
T/F: Detecting Inhibitors
Internal positive controls (IPC)
o PCR: 16S is the housekeeping gene
T
T/F: Overcoming Inhibitors should be removed during DNA purification
T
is a strand of short nucleic acid sequences
that serves as a starting point for DNA synthesis.
Primer
It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA.
Primer
T/F: The polymerase starts replication at the 3’-end of the primer, and copies the opposite strand.
T
T/F: Target sequence and designing primers substantially affect the efficiency of your PCR
T
Primer melting temperature range
50C-65C
T/F: GOOD PRIMER’S CHARACTERISTIC
Absence of dimerization and hairpin capability
T
T/F: The presence of G or C bases within the last five bases from the 3’ end of primers (GC clamp) to enhance annealing of the end which will be extended due to the stronger bonding of G and C bases. More than 3 G’s or C’s should be avoided in the last 5 bases at the 3’ end of the primer – mispriming
T
TARGET SEQUENCE FOR PCR
Conventional PCR
200-800 bp (~500)
TARGET SEQUENCE FOR PCR
Real Time PCR
75-200 bp (~100)
T/F: Short PCR products are typically amplified with higher efficiency than longer ones; but should be at least 75 bp to easily distinguish from any primer-dimers
T
is an oligonucleotide sequence – will target a specific sequence of opposite base pairing (A-T, G-C only) of single stranded nucleic acids
Primer
PRIMER SPECIFICITY
amplifies ALL bacterial DNA for instance
universal
PRIMER SPECIFICITY
amplify all denitrifies for instance
group specific
PRIMER SPECIFICITY
amplify just a given sequence
specific
T/F: PRIMER UNIQUENESS
There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA, avoiding the possibility of mis-hybridization to a similar sequence nearby.
T
T/F: PRIMER UNIQUENESS
There shall be an annealing site in possible
contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome)
F; no annealing site
T/F: PRIMER UNIQUENESS
o the longer the primer, the more chance that it is unique;
o the longer the primer, the higher melting/annealing temperature – specificity
T
The length of primer has to be at least __ bases to ensure uniqueness
15 bases
Tm is characteristics of the DNA/Base composition; Higher G+C content DNA, has a higher Tm due to more _______
hydrogen bonds
T/F: Above 30 bases of primers has a risk of mispairing, primer dimers and hairpin
T
used for oligonucleotides with short sequences lengths, i.e. those that are 14 bases or less
assumes a primer concentration of 50 nM, a monovalent (Na+) ion concentration of 50 mM, and pH 7.0
Basic Method (Marmur Doty formula)
T/F: LIMITATION OF BASIC METHOD
Marmur and Wallace formula Tm estimation only take into account the number of GC and AT nucleotides
T
↑ Ta = Insufficient primer-template hybridization = _ PCR product yield
↓
↓Ta = Non-specific products cause by _ base pairs
↑
T/F: ANNEALING TEMPERATURE
If primers can anneal to themselves or anneal to each other (primer dimer) rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided.
T
can be added in the same tube amplify multiple sites
multiplex PCR
Design difficulty
o Similar melting Temperature
o No dimer formulation (cross-dimer)
o The products need to be of different sizes if visualization by gel – or use different probes/fluophores
multiplex PCR
Primers can also be designed to amplify multiple products - “universal primers”.
multiplex PCR
Application example of Multiplex PCR
Genome Identification
is the enzyme responsible for copying
the sequence starting at the primer from the single DNA strand
DNA Polymerase
This enzyme is heat-tolerant → useful both because it is thermally tolerant (survives the melting T of DNA denaturation) which also means the process is more specific, higher temps result in less mismatch – more specific replication
DNA Polymerase
DNA POL ALTERNATIVES
no proofreading activity. It is high in fidelity and less error.
Pyrococcus furiosus (Pfu polymerase)
DNA POL ALTERNATIVES
Tth polymerase
Thermus thermophilus
DNA POL ALTERNATIVES
Tfl polymerase
Thermus flavus
DNA POL ALTERNATIVES
Tli polymerase aka Vent polymerase
Thermococcus litoralis
DNA POL ALTERNATIVES
Deep Vent polymerase
Pyrococcus species GB-D
The standard here is Taq polymerase, which has a ________ of 50-60 nucleotides (nt) per second at 72C
Processivity
refers to the accuracy of the complementary copy being made
Fidelity
has among the highest error rates
of the thermophilic polymerases at 285 x 10-6 errors per template nucleotide
Taq DNA polymerase
has a proofreading ability that is five-fold
better than Taq at 57 x 10-6 errors per template nucleotide and Pfu polymerase also demonstrates fidelity in this range
Tli polymerase
refers to the stability of the enzyme at high temperature, is intimately linked to the other two polymerase attributes
Persistence
Stability can be measured in terms of how long the enzyme retains at least one-half of its activity during sustained exposure to high temperature.
Persistence
is the reagent of choice for most PCR
amplifications and the best choice for conventional PCR
o Average size of amplicon: >500 BP
Taq polymerase
T/F: Taq polymerase is an optimum choice for DNA sequencing
F; not an optimum
T/F: ADVANTAGE OF TAQ POLYMERASE
DNA polymerases from various species of the genus Thermus have a very unusual property not shared by other DNA polymerases
T
T/F: ADVANTAGE OF TAQ POLYMERASE
These enzymes possess 3’→5’ proof reading ability
F; These enzymes do not possess 3’→5’ proof reading ability whereas other polymerases do possess this ability
T/F: ADVANTAGE OF TAQ POLYMERASE
The consequence of the lack of 3’→5’ proof reading ability is that Taq polymerase adds a single 3’ nucleotide (Adenosine) on both strands of every amplicon
T
Cofactor required for activation of Taq Polymerase
Mg2+ in PCR
T/F: ADVANTAGE OF TAQ POLYMERASE
This 3’ extension permits direct cloning of a PCR product using one of the various commercially available PCR cloning vectors
T
T/F: Each PCR reaction has an optimal concentration of free Mg2+
T
dNTP’s bind free Mg2+ in a ____ molar ratio
1:1
T/F: Increases or decreases in [dNTP] must be matched with equivalent changes in [Mg2+]
T (1:1 molar ratio)
- Stabilizes the DNA polymerase, DNA, and nucleotides
- 500 mM KCl
- 100 mM Tris-HCl, pH 8.3
- Triton X-100 or Tween
Buffer
- Contains region to be amplified
- Any DNA desired
- Purity not required
- Should be free of polymerase inhibitors
DNA template
- The medium for all other components.
- Specially purified, double distilled, deionized, autoclaved, nuclease-free, and does not contain detectable amounts of nucleic acid
Water
- Added to the growing chain
- Activated NTP’s
- dATP, dGTP, dCTP, dTTP
- Stored at 10mM, pH 7.0
- Add to 20-200 uM in assay
Nucleotides
- Specific for ends of amplified region
- Forward and Reverse
- Annealing temps should be known
- Depends on primer length, GC content, etc.
Length 15-30 nt
Conc 0.1 – 1.0 uM (pMol/ul)
Primers
- Essential co-factor of DNA polymerase
- Too little: Enzyme won’t work.
- Stabilizes the DNA double-helix
- Too much: DNA extra stable, non-specific
priming, band smearing - Used at 0.5 to 3.5 uM in the assay
Mg++ ions
- The enzyme that does the extension
- TAQ or similar
- Heat-stable
- Approx 1 U / rxn
DNA Polymerase
DNA amplified is known as
amplicon
T/F: The shorter the DNA base pair, the faster it will migrate from cathode to anode.
T
T/F: If (+) control and a band were detected: the control has a contaminant
T
T/F: If (-) control is added and it results in no band, it means that the DNA amplified is successful and there are no contaminants; you can also conclude that the technique is almost perfect.
T
Used in Convention and real-time platform
o Conventional is also called as end point that needs electrophoresis to detect the amplification.
Nested PCR
was developed to increase both the sensitivity and specificity of PCR
Nested PCR
2 primers and run in 30 cycles
Nested PCR
The products of the first round of amplification are then subjected to a second round of amplification using the
second set of primers. The second set of primers anneal to a sequence internal to the sequence amplified by the first primer set.
Nested PCR
This technique uses two pairs of amplification primers and two rounds of PCR
Nested PCR
T/F: NESTED PCR
The increased sensitivity arises from the high total cycle number, and the increased specificity arises from the annealing of the second primer set to sequences produced by the second round.
F; first round
Major concern of Nested PCR
contamination that occurs during the transfer of the first-round product to the second tube for the second round of amplification
How to avoid contamination in nested PCR?
physically separating the first- and second-round amplification mixtures with a layer of wax or oil
Amplifying the different genes simultaneously
Multiplex PCR
two or more primer sets designed for
amplification of different targets are included in the same PCR reaction
Multiplex PCR
Using this technique, more than one target sequence in a clinical specimen can be amplified in a single tube
Multiplex PCR
T/F: MULTIPLEX PCR
The amplicon sizes should be different enough to form distinct bands when visualized by gel electrophoresis
T
can be designed in either single-template
PCR reaction that uses several sets of primers to amplify specific regions within a template, or multiple-template PCR reaction, which uses multiple templates and several primer sets in the same reaction tube
Multiplex PCR
can reduce costs and time to simultaneously detect two, three, or more
pathogens in a specimen, it is more complicated to develop and often is less sensitive than single-primer-set PCR.
Multiplex PCR
The advantage of it is that a set of primers
can be used as internal control, so that we can eliminate the possibility of false positives or negatives
Multiplex PCR
can save costly polymerase and template
in short supply
Multiplex PCR
transforms the exponential data from
conventional PCR to digital signals that simply indicate whether or not amplification occurred
Digital PCR
is accomplished by capturing or isolating each individual nucleic acid molecule present in a sample within many chambers, zones, or regions that are able to localize and concentrate the amplification product to detectable levels
Digital PCR
has many applications, including detection
and quantification of low levels of pathogen sequences, expression of rare genetic sequences in single cells, and
clonal amplification of nucleic acids for sequencing mixed nucleic acid samples
Digital PCR
The capture or isolation of individual nucleic acid molecules may be done in capillaries, microemulsions, or arrays of miniaturized chambers, or on surfaces that bind nucleic acids
Digital PCR
