NUCLEIC ACID AMPLIFICATION
Nucleic Acid Amplification
Three Categories:
1. Target amplification systems
2. Probe amplification systems
3. Signal amplification systems
TARGET AMPLIFICATION
- TARGET is a short region of double-stranded DNA
- Involves making many copies of a specific DNA
sequence
- PCR is the first and prototypical method for amplifying
target nucleic acid
- Discovered by Kary Mullis in 1983
POLYMERSE CHAIN REACTION
- The first successful amplification was a short
fragment of the Escherichia coli plasmid (pBR322)
- The first practical application is the amplification of
beta-globin and analysis for diagnosis of patients with
sickle cell anemia
- All use enzyme-mediated processes, to synthesize
copies of target nucleic acid
- Amplification products detected by 2 oligonucleotide COMPONENTS:
primers 1. DNA template
- Produce 108-109 copies of targeted sequences - It will be coming from the sample sequence that
(AMPLICONS) we have.
- Sensitive to contamination à false-positive reaction - EX. SARS-COV2 detection
- 4 types of deoxynucleotide or dNTP’s - Nasopharyngeal/oropharyngeal/ swab or
(deoxynucleotide triphosphate) saliva serves as the sample to detect for the
• dATP presence of RNA.
• dTTP REMEMBER! PCR requires DNA. That is why we have
• dCTP modifications of PCR: RT-PCR, RT-q-PCR [Reverse
• dGTP Transcriptase-quantitative-PCR/ real time (combination
- Polymerase enzyme – number one enzyme that are
using. • Real-Time: quantitative PCR
- denatured/separate the DNA, it requires high • RT-PCR: Reverse Transcriptase
temperature (90-92°C) 2. Short oligonucleotide primers
• 2 types of Primer: Forward and Reverse
(depending if template or non-template strand)
AMPLIFICATION PROGRAM • REMEMBER! dsDNA is Antiparallel
Components: 3. Nucleotides
1. DNA template - These are the dNTPs (deoxynucleotide
2. Short oligonucleotide primers triphosphate) which has 3 phosphates and is
3. Nucleotides being used both vitro and in-vivo
4. Polymerase 4. Polymerase
5. Buffers 5. Buffers
- Must maintain the correct pH because any
changes affect the amplification procedure
- PCR is pH sensitive
- Enzyme is pH specific (wrong pH, no reaction/
catalyzation to new strand will occur)
MgCl2: cofactor of enzyme to catalyze the adhesion of
dNTPs to growing strand of DNA
Elements of PCR cycle MELTING TEMPERATURE (Tm) formula:
1. DENATURATION
2. ANNELING Tm= 4°C x number of GC pairs + 2°C x number of AT
3. EXTENSION/ELONGATION pairs
Wherein,
Tm= 4°C x number of (Guanine + Cytosine) + 2°C x
number of (Adenine + Thymine)
PRIMER EXTENSION
- Last step in the first cycle only
- the polymerase synthesizes a copy of the template
DNA by adding nucleotides to the hybridized primers
- DNA polymerase replicates the template DNA by
simultaneously extending the primers on both strands
of the template
DENATURATION - This step occurs at the optimal temperature of the
- First step in amplification program enzyme 68°C to 75°C
- Separation from double to single strand to be used as - Polymerase enzyme will be utilized here
template. • It will catalyze the adhesion of DNTPs to have the
- Both forward are reverse are used as template new strand of DNA or the amplicon.
- In-vivo replication (both leading and lagging start are • It is very specific and must be complementary
being used to replicate DNA) dsDNA→ ssDNA o Adenine- thymine
- In PCR we are requiring dsDNA (Double Stranded o Guanine- Cytosine
DNA) - In this step, the polymerase synthesizes a copy of the
- Remember that: RNA is ssDNA and are easily template DNA by adding nucleotides to the hybridized
denatured by high temperature but dsDNA will be primers
denatured in a way that will be separated only without - DNA polymerase replicates the template DNA by
disrupting the structure simultaneously extending the primers in both strands
- (Ranges from 90-95C but not more than 95 of the template.
- Genomic DNA (longer template) - This step occurs at the optimal temperature of the
- A-T has 2 H bonds enzyme 68°C to 75°C.
- G-C has 3 H bonds - The primer will be elongated by the enzyme
PRIMER ANNEALING Tm= 4°C x number of GC pairs + 2°C x number of AT
- Most critical for the specificity of the PCR pairs
- The primers dictate the part of the template that will
be amplified Tm= 4°C x number of (Guanine + Cytosine) + 2°C x
- Position primer to the sequence number of (Adenine + Thymine)
- Annealing temperatures range from 50°C to 73°C
- A starting point can be determined using the Tm
(MELTING TEMPERATURE) of the primer
sequences
- The Tm is a way to express the amount of energy
required to separate the hybridized strands
of a given sequence
- Annealing Temperature: temperature wherein the
primer will bind to the target sequence
- Melting temperature: temperature wherein primer
dissociates/ denature/separate
- Primers are oligonucleotides which are very short (18-
25 sequence or 18-30)
AMPLIFICATION BY PCR MELTING TEMPERATURE
- Temperature wherein the double stranded helix or
PCR: First Four Cycles sequence of primer will start to dissociate or separate.
- Number of the double strands with the right length; - Primer Tm then serves as a starting point for setting
exact site will only be amplified. the optimal annealing temperature (below 5°C)
• 1st cycle: 1 dsDNA Tm = 4°C × number of GC pairs + 2°C × number of AT
• 2nd Cycle: 2 dsDNA pairs
- EXPONENTIAL AMPLIFICATION- the exact size only - Forward primer Tm= Reverse primer Tm
is amplified. - The melting temperature (Tm) of
- During the 35th Cycle, how much is amplified? - Forward (F) = 60 Celsius
- 2 = 34 billion copies; this only took 2 hours. If in vivo - Revers (R)= 58 Celsius
it will take weeks or months to amplify. So, this is the o Therefore, the Annealing Temperature (Ta) is:
purpose why we do PCR amplification. Forward (F) = 55 Celsius
- 2 PURPOSES OF AMPLIFICATION TECHNIQUE: o Revers (R)= 53 Celsius
1. To make copies
- Used for preparation for certain tests PROBLEMS ECOUNTERED IN ANNEALING
2. For detection 1. Mispriming
- Diagnosis if present of absent - The primer binds to the wrong sequence
- Unintended priming or adding in the sequence;
priming a different sequence.
PCR: Completed Amplification Cycle *Remember: in gel electrophoresis we are basing on the
size of the Amplicons. If the size of Amplicons changed
Components of PCR and became longer; the migration in the gel
1. DNA template electrophoresis will change as well. Hence, changing
2. Short oligonucleotide primers interpretation.
3. Nucleotides Remember: Primer is the key for specificity of PCR;
4. Polymerase • The size of the primer should be 18-30 Base Pairs
5. Buffers - If used shorter than 18: it can cause mispriming
because it can have many identical short
1. PRIMERS sequences.
- PROBES: bind directly beside the target sequence - If shorter= prone to mispriming
- PRIMERS: bind/ complementary to the sequences
near the target sequence. PRIMER DIMER
- Primers are designed to contain sequences - Binding of primers onto each other through short (2-
complementary to sites flanking the region to be to 3-base) homologies at their 3ʹ ends and the
analyzed copying of each primer sequence
- Single-stranded DNA fragments, usually 20-30 bases
in length NON-COMPLEMENTARY EXTENSIONS
- The placement of the primers will also dictate the size - Designed to add or alter sequences to one or both
of the amplified product ends of the PCR product
- - Also known as: Tail
- Added in the 5’ end of the primer
Two Types of Primer - Also called as: Overhangs
1. Forward Primer - Purpose: Placing of labels: Biotin or fluorescent dyes
- Binds to the template strand or the Minus Strand.
- 5’→ 3’ 2. DNA Template
2. Reverse Primer - Genomic DNA will have only one or two copies per
- Binds to the non-template strand or Plus Strand. cell equivalent of single-copy genes to serve as
- 3’→ 5’ amplification targets
- For routine clinical analysis, 100 ng to 1 ug of DNA
5’ = has the phosphate group is usually used
3’ = free sugar group - Good condition, free of contaminating proteins, and
without nicks or breaks that can stop DNA synthesis
or cause misincorporation of nucleotide bases
Sources: 4.A.Modified Polymerase Enzymes
• Patient’s genomic or mitochondrial DNA 1. Stoffel fragment
• Viruses - Modified version of Taq polymerase
• Bacteria - Lacking the 289 N-terminal amino acids of Taq
• Fungi polymerase
• Parasites - half-life of the Stoffel fragment at high
temperatures is about twice that of Taq
3. Deoxyribonucleotide Bases polymerase
- Equimolar mixture of the four deoxynucleotide - recommended for allele-specific PCR and for
triphosphates (dNTPs) amplification of regions with high GC content
- Standard procedures require 0.1 to 0.5 mM - ddNTP (Dideoxynucleotide triphosphate) used
concentrations of each nucleotide in DNA Sequencing
- 4 Different dNTPs: 2. ThermoSequenase
• Adenine 3. T7 Sequenase
• Thymine
4.B. THERMOSTABLE POLYMERASE ENZYMES
• Cytosine
• Guanine
4. DNA Polymerases
- First discover in bacteria
- similar to the nomenclature of restriction enzyme.
WHY E. COLI IS NOT SUCCESSFUL?
- because of the temperature: it is heat labile
- (90-95°C)
- They successfully retried bacteria from volcanic
craters they successfully got a thermophilic
bacteria called Thermus aquaticus (most
common DNA polymerase to be used)
- It has a capability to resist high temperature, the
polymerase enzyme of that bacteria is stable at
high temperature, that is why they used it for
polymerase chain reaction - Extension rate (Nucleotide/Sec): Taq pol can add
1. Taq polymerase 75 dNTPs per second to the growing strand of DNA
- Thermostable enzyme
- Isolated from the thermophilic bacterium 5. PCR BUFFERS
Thermus aquaticus - Provide the optimal conditions for enzyme activity
- DNA polymerase added once at the beginning of (8-9 ph)
the procedure and would maintain its activity - Uses monovalent cations (e.g KCl (Potassium
throughout the amplification cycles. Chloride), (NH4)2SO4 (Ammonium Sulfate) and
2. Tth polymerase divalent cations such as MgCl2 (Magnesium
- From Thermus thermophilus chloride)
- HAS MODIFICATION: Has reverse- - Each NTP will take up one magnesium atom
transcriptase - The recommended range of MgCl2 concentration is
- activity, 1 to 4 mM in standard reaction conditions
- Has reverse-transcriptase activity can be used in
reverse transcriptase PCR (RT-PCR) *Too few Mg2+ ions → lower enzyme efficiency à low
yield of PCR product
3.*Vent polymerase (Proofreading enzyme) *Overly high Mg2+ → promote misincorporation à
- Allows Taq or Tth polymerase to generate large increase the yield of nonspecific products
products over 30,000 bases in length.
Remember: EDTA is a chelator of Calcium and
Magnesium. If the sample contains EDTA, the magnesium
chloride concentration should be adjusted accordingly.
If has EDTA= Increase the Magnesium Chloride
concentration
o Commercially available and bought
Other components o Added to know the size of the fragments
• Tris buffer and additional buffer components o Known size; serves as a control
- to maintain the pH of the buffer - Last gel lane= Reagent Blank
- Optimal pH for PCR: 8-9.5 o Doesn’t have any reagents. Expected to do not
• 10 mM Tris-HCl have the presence of bonds. Since there is no
- maintain the pH; preventing the over reagent, only the sample. No products
alkalinization of buffer o If there is a band. It means that there is a
• Bovine serum albumin (10 to 100 μg/ mL) contamination.
- Stabilizes the enzyme, binding to the inhibitors o Reject the result. If there is a band in the
• Dithiothreitol (0.01 mM) reagent blank.
- enhance enzyme activity o PCR products after resolution on a gel
• Formamide (1% to 10%) o The size, presence, or intensity of PCR
- lowers the denaturing temperature of the DNA to products is observed after electrophoresis
avoid many secondary structures
• Chaotropic agents (detergents) such as Triton X- CONTROLS FOR PCR
100, glycerol, and dimethyl sulfoxide 1. Positive Control
- Expected to have a band
THERMAL CYCLER/ THERMOCYCLERS - Ensure that the enzyme is active, the buffer is
- 96 wells (more common): 96 tubes can be placed : optimal, the primers are priming the right
0.2 mL sequences, and the thermal cycler is cycling
- 384 wells: 0.04 mL appropriately.
- has the capability to change the temperature of the 2. Negative control
environment for short period of time. a. Without DNA
- Designed to rapidly and automatically ramp o Also called as contamination control or reagent
(change) to the required incubation temperatures, blank
holding at each one for designated periods o Ensures that the reaction mix is not contaminated
- Designed with heated lids that eliminated the with template DNA or amplified products from a
requirement for vapor barriers previous run
- Real-time PCR systems are equipped with b. With DNA that lacks the target sequence
fluorescent detectors to measure the PCR product o Also called as negative template control
as the reaction proceeds o Ensures that the primers are not annealing to
- Conventional PCR are Qualitative (checking the nontarget sequences of DNA
presence or absence of target sequence) o Primer is non-specific or not optimal; they bind to
- Real-time PCR or qPCR (Quantitative PCR) non-target unintended sequence.
THE REACTION IN PCR CONTAMINATION OF PCR REACTIONS
- Preparation of the specimen for PCR is classified as - Very common contamination: Open tube method
a pre-PCR procedure - Any molecule of DNA containing the intended target
- PCR tube is really small and has a capacity of 0.2ml sequence is a potential source of contamination
or 200 ul - The most dangerous contaminant is PCR product
- The final volume of the reaction mix varies from 1 to from a previous reaction
50 μL loaded into the thermal cycler programmed - Laboratories are designed to prevent exposure of pre-
with the temperatures and times for each step of the PCR reagents and materials to post-PCR
PCR cycle contaminants
- Thermal cyclers take thin-walled tubes, tube strips, - Contamination control procedures, therefore, are
or 96-well or 384-well plates with 0.04- to 0.2-mL mainly directed toward eliminating PCR product from
volume capacity the setup reaction
- The number of cycles to be completed usually is 30
to 50 cycles.
- PCR products after resolution on a gel
- The size, presence, or intensity of PCR products is
observed after electrophoresis
- First gel lane= Molecular-Weight Marker (Ladder)
o Found: 1st gel lane.
o Known sizes of fragments
CONTAMINATION CONTROL TOUCHDOWN PCR
1. Physical - Modification of the PCR program used to enhance the
- Separation of Pre- PCR and Post- PCR areas amplification of the desired PCR product
- Air-locks, positive air flow (push airflow) - Annealing temperatures set higher than the optimal
- PCR hoods with UV light target primer- binding temperature
o UV Light: (common room decontaminant - Decreased by 1°C every cycle
to maintain the Pre-PCR area)
o Purpose: interfere with the replication. It PCR PRODUCT CLEAN-UP
catalyzes the single and double strand. 1. Gel elution
Breaking the DNA and RNA for it not to be - Removes all reaction components as well as
amplified. misprimes and primer dimers
o The effectiveness of UV Light can be - enzyme used: B- Agarase or iodine
modified. Increased. Adding Psoralen 2. Solid phase isolation of PCR product
o Psoralen: Added to the actual - Amplicons free of PCR components are
amplification product after analysis. It conveniently prepared using spin columns or silica
intercalates between the dsDNA and beads.
when exposed to UV light with its long - Remove the residual PCR mix: primer, salt, dNTPs
waves. It will add to the Thymidine, 3. Enzymatic method
Cystidine, Uracil - Addition of alkaline phosphatase (AP) in
- Prevent the amplification: UV Light and Psoralen combination with exonuclease I (ExoI) is an
2. Chemical /Enzymatic enzymatic method for removing nucleotides and
- dUTP + uracil-N-glycosylase (added to the PCR primers from PCR products prior to sequencing or
reaction) mutational analyses.
- Called as the dUTP UNG system during the - Alkaline phosphatase (AP): for
reagent master mix dephosphorization of unincorporated
- Psoralen + UV (depends on UV wavelength and nucleotides. We have extra dNTPs (inactivated).
distance to surface) - Exonuclease I (ExoI): Remove/degrade Single
- 10% bleach (most effective for surface stranded primers. Also, extra or unused primers.
decontamination)
3. Wipe Test
- Filter paper is wiped on any exposed or touched
surfaces in the pre-PCR setup, extraction, and
amplification areas
MISPRIMING
- When PCR products are analyzed for size and purity
by electrophoresis, the amplicon size should agree
with the size determined by the primer placement
- Could be averted by good primer design and optimal
amplification conditions
- Occur during the preparation of the reaction mix (Taq
Polymerase) and could be prevented by using Hot-
Start PCR
HOT START
Hot-start PCR can be performed in three different ways:
1. Prepare reaction mixes on ice
2. Place in preheated cycler
3. Use a sequestered enzyme that requires an initial heat
activation.
• Platinum Taq
• AmpliTaq Gold
• HotStarTaq
