4. NUCLEIC ACID AMPLIFICATION - LAB Flashcards
make many copies of target DNA
NUCLEIC ACID AMPLIFICATION
Three Categories of Nucleic acid amplification
- Target amplification systems
- Probe amplification system
- Signal amplification system
category of NAA that targets nucleic acid based (PCR)
Target amplification systems
category of NAA that is probe specific for target sequence
Probe amplification system
is a short region of double-stranded DNA
TARGET
Involves making many copies of a specific DNA sequence
TARGET AMPLIFICATION
is the first and prototypical method for amplifying target nucleic acid
PCR
PCR Discovered by
Kary Mullis in 1983 (Nobel Prize Awardee)
Involves making copies of a target sequence to such a level (in the millions of copies) that they can be detected in vitro (be visualized on an agar plate).
Target Amplification
PCR
The first successful amplification was a short fragment of the
Escherichia coli plasmid (pBR322
The first practical application is the amplification of amino acid
sequence of beta- globin and analysis for diagnosis of patients with
sickle cell anemia
PCR products
Amplicons
binds beside the target sequence
Primer
binds directly to the target sequence
Probe
AMPIFICATION PROGRAM Components:
DNA template
Short oligonucleotide primers
Nucleotides
Polymerase
Buffers
Short oligonucleotide primers types
FORWARD AND REVERSE
useful in addition of dNTPs to growing strands
Polymerase
maintain and stabilize condition→ pH (stabilize enzyme) and Mg, NaCl for temp
Buffers
ELEMENTS OF PCR CYCLE
- Denaturation
- Annealing
- Extension/Elongation
Denaturation Temperature (°C)
Time (sec)
90-96 temp
20-60 time
Annealing Temperature (°C)
Time (sec)
50-70 temp
20-90 time
Extension/Elongation Temperature (°C)
Time (sec)
68-75 temp
10-60 time
always bind to the minus strand or anti-sense strand (TEMPLATESTRAND)
Forward Primer
Forward Primer formation of amplicons
Forward from 3’ to 5
binds to plus strand or sense strand (NON-TEMPLATE)
Reverse Primer
Reverse Primer formation of amplicons
3’ to 5’ pero baliktad kasi yung strand kaya reverse ok????
Tm formula for annealing
Tm = 4x #GC + 2x#AT
attachment of primer away from the target sequence
Mispriming
Binding of primers onto each other through short (2- to 3-base)
homologies at their 3’ ends and the copying of each primer sequence
Primer Dimer
DNA TEMPLATE Sources:
- Patient’s genomic or mitochondrial DNA
- Viruses
- Bacteria
- Fungi
- Parasites
DNA POLYMERASES used in PCR
Taq polymerase
Tth polymerase
Vent polymerase
- most known polymerase for PCR
- Thermostable enzyme (stable even at high temp)
Taq polymerase
Taq polymerase Isolated from the thermophilic bacterium
Thermus aquaticus
Tth polymerase From
Thermus thermophilus
Has reverse-transcriptase activity, so it can be used in reverse transcriptase PCR
Tth polymerase
Allows Taq or Tth polymerase to generate large products over 30,000 bases in length.
- exonuclease, removing non-complementary and unnecessary bases, exons especially for very long DNA bases
Vent polymerase
Proofreading enzyme
Vent polymerase
- Lacking the 289 N-terminal amino acids of Taq polymerase
recommended for allele- - specific PCR and for amplification of regions with high GC content
Stoffel fragment
Modified Polymerase Enzymes
Stoffel fragment
Accessory components
- Bovine serum albumin (10 to 100 µg/mL)
- Dithiothreitol (0.01 mM)
- Formamide (1% to 10%)
- Chaotropic agents (detergents) such as Triton X-100, glycerol, and dimethyl sulfoxide
Accessory components for the stabilization of enzyme
- Bovine serum albumin (10 to 100 µg/mL)
Accessory components to enhance the enzyme activity
- Dithiothreitol (0.01 mM)
Accessory components lower denaturation temperature to reduce secondary structure
Formamide (1% to 10%)
Accessory components for stability of enzyme and reduce secondary structure
Chaotropic agents (detergents) such as Triton X-100, glycerol, and dimethyl sulfoxide
Designed to rapidly and automatically ramp (change) to the required incubation temperatures, holding at each one for designated periods
- Designed with heated lids that eliminated the requirement for vapor barriers
THERMAL CYCLER/THERMOCYCLERS
(Quantitative)systems are equipped with fluorescent detectors to measure the PCR product as the reaction proceeds
Real-time PCR
Qualitative presence or absence of amplicons
Conventional PCR- End point Analysis
CONTROLS FOR PCR
Positive Control
Negative control
Ensure that the enzyme is active, the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately
Positive Control
- Also called as contamination control or reagent blank
- Ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run
Negative control
Without DNA
- Also called as negative template control
- Ensures that the primers are not annealing to nontarget sequences of DNA
Negative control With DNA
CONTAMINATION CONTROLS for PCR
Physical
Chemical or enzymatic
Wipe test
- Separation of Pre- PCR and Post- PCR areas
- Air-locks, positive air flow, one way
- PCR hoods with UV/ biosafety cabinet
Physical contamination control
- dUTP + uracil-N-glycosylase (added to the PCR reaction)- instead of adding dNTP, add dUTP = UNG (Uracil N-glycosylase) will degrade all DNA with uracil
Chemical/Enzymatic contamination control
most effective for surface decontamination
10% bleach
will intercalate in DNA, resist amplification
Psoralen
- Filter paper is wiped on any exposed or touched/ contaminated surfaces in the pre- PCR setup, extraction, and amplification areas
Wipe Test
Used to remove the possible mispriming
Hot start
Hot start 3 ways
on ice
preheated cycler
sequestered enzyme
