NUCLEIC ACID AMPLIFICATION Flashcards
- Most common nucleic acid amplification method
POLYMERASE CHAIN REACTION
What the 3 categories in Nucleic acid amplification?
TARGET AMPLIFICATION
PROBE AMPLIFICATION
SIGNAL AMPLIFICATION
Advantages of PCR:
User-friendly, more automated, and more amenable to use
o Copy of the target DNA
o Products of PCR
o Multiple copies produced
AMPLICONS
Discovered in 1983 by Kary Mullis
o Escherichia coli plasmid pBR322 (1st to be amplified)
o Method was called “polymerase-catalyzed chain reaction”
PCR
- 1st to be amplified
Escherichia coli plasmid pBR322
- Application of heat to separate dsDNA (template) into 2 single strands
- Denatured by heat
▪ Temperature is enough to break hydrogen bonds
DENATURING (90-96C)
▪ Temperature is enough to break hydrogen bonds
▪ Application of heat to separate dsDNA (template) into 2 single
strands
DENATURING
DNA TEMPLATE is derived from?
▪ Patient’s genomic DNA
▪ Patient’s mtDNA
▪ DNA from viruses, bacteria, parasites
90-60 CELSIUS
DENATURING
50-70 CELSIUS
ANNEALING
Binding of primers to ssDNA template by hybridization
▪ Most crucial step
▪ 50oC to 70oC, 20sec – 90 sec
ANNEALING
Primers are also known as?
OLIGODEOXYNUCLEOTIDES
This step determine the specificity of PCR.
ANNEALING
-More specific & hybridize at slower rate
LONG PRIMERS
- Less specific & hybridize at faster rate
SHORT PRIMERS
2 TYPES OF PRIMERS:
FORWARD , REVERSE
- Artifact composing of 2 primers binding onto each other
- Double the size of the utilized primer
- Occur where there are 3 or more complementary happens at 3’ end of primers
PRIMER DIMERS
(68oC - 75oC)
ELONGATION/EXTENSION
- Aberrant primer binding
- Primers bind to unintended sequences
MISPRIMING
WHAT ARE THE FACTORS THAT MAY AFFECT THE EXACT Tm
- Reactive conditions
- Salt concentrations
- Template conditions
- Secondary structure (internal folding & hybridization between DNA
strands)
DNA polymerase helps here but should be thermostable
o Obtained from bacteria (thermophilic bacteria)
▪ DNA synthesis occurs
▪ Addition of nucleotides (primer extension)
EXTENSION/ELONGATION
10sec – 60sec
EXTENSION/ELONGATION
20-60 secs
DENATURING
20-90 secs
APPEALING
- Enzyme for the addition of nucleotides to the hybridized primer
- High temperature in denaturation step will inactivate the enzyme
DNA POLYMERASE
TYPES OF DNA POLYMERASE
➢ Mainly utilized in PCR
➢ Obtained from Thermus aquaticus
➢ Thermostable
➢ By Randall Saiki
➢ Commonly used
Taq POLYMERASE
Taq POLYMERASE is obtained from?
- THERMOSTABLE
THERMUS AQUATICUS
TYPES OF DNA POLYMERASE
➢ Utilized in reverse-transcriptase PCR (RNA)
➢ Obtained from Thermus thermophilus
Tth POLYMERASE
Tth POLYMERASE is obtained from?
Thermus thermophilus
TYPES OF DNA POLYMERASE
➢ Tli polymerase
➢ Allows Taq or Tth to generate large products
(30,000 bases)
➢ Obtained from Thermococcus litoralis
VENT POLYMERASE
VENT POLYMERASE is obtained from?
Thermococcus Litoralis
- Incorporates ddNTPS
- Chain termination sequencing
THERMOSEQUENASE AND 17 SEQUENASE
MODIFIED TAQ VERSIONS:
- 2x the half-life of Taq in high temperature
- Broader MgCl2 concentrations
- For allele specific PCR and Amplification of GC content
STOFFEL FRAGMENT
Why is vigorous agitation of enzyme is not recommended?
- may alter the structures of enzyme and will denature irreversibly and air bubbles may damage
it
PCR REAGENTS MASTER MIX:
buffer + DNA template + DNA polymerase + dNTPs (deoxyribonucleotide triphosphates) + KCl + MgCl + Primers
- It Directs DNA synthesis to the desired region
PRIMERS
- Divalent cation, required by the enzyme
1.5 mM MgCl2
Building blocks that extend the primers
dATP, dCTP, dGTP, dTTP (dNTPs)
Buffer to maintain optimal pH (8-9.5) for the enzyme reaction
Tris, pH 8.4
- Monovalent cation (salt), for optimal hybridization of primers to template
KCl
- A mixture of 4 equimolar deoxynucleotide triphosphates
- 0.1-0.5 mM concentration of each
- Concentration and purity may affect PCR results
- High concentrations (10-100 mM) are recommended for storage to
present breaking down to dNDP & dMNP = inhibit PCR - dNDP(diphosphate) & dMNP (monophosphate) can also be a result
of poor manufacturing and contamination of heavy metals
DEOXYRIBONUCLEOTIDE BASES
- provide optimal conditions for enzyme activity (pH 8-9.5)
- increased salt concentration may denature long DNAs slower than short DNAs
PCR BUFFERS
▪ Lower denaturing temperature
Formamide (1% - 10%)
PCR EQUIPMENT
o 1st PCR system conducted
o Replaced by automated methods and thermostable
enzymes
WATER BATHS & HEAT BLOCKS
PCR EQUIPMENT
- For rapid and automatically ramp to the required temperatures
THERMAL CYCLERS/THERMOCYCLERS
PCR products can be analyzed by:
o Gel/capillary electrophoresis
o Nucleic acid analysis
o Other molecular biology tests
PCR CONTROLS
- AKA contamination control / reagent blank
- Lacks DNA template
NEGATIVE CONTROLS
PCR CONTRTOLS
- Ensure
o Enzyme is active
o Buffer is optimal
o Primers are priming right sequences
o Thermal cycles are cycling properly
POSITIVE CONTROLS
PCR CONTROLS
- With a DNA template that has no target sequence
- There should be no annealing
NEGATIVE TEMPLATE CONTROLS
PCR CONTROLS
- Contain a primer binding site
- Determine false negative (amplification failure)
AMPLIFICATION CONTROLS
- Common in open tube methods
- Most common sources:
o Products of previous amplifications (may aerosolized if
uncapped)
PCR CONTAMINATION
WHAT IS THE MOST COMMON SOURCE OF PCR CONTAMINATION?
Products of previous amplifications (may aerosolized if
uncapped
CONTROL METHODS
o 10% bleach (7 mM sodium hypochlorite)
* For decontamination of workspaces
o Enzymatic method
* UNG for previous amplifications (enzyme: uracil-Nglycosylase)
o Wipe tests
* To determine concentration
CHEMICAL CONTAMINATION CONTROL
CONTROL METHODS
o Separate pre- & post-PCR areas
o Positive airflow, airlocks
o Equipment, PPE, & reagents should be dedicated to either
pre- or post-PCR
o UV for decontamination
o Psoralens
o Attach T, U, C, in DNA under UV (prevent
denaturation & amplification)
PHYSICAL CONTAMINATION CONTROL
CONTROL METHODS
* For decontamination of workspaces
10% bleach (7 mM sodium hypochlorite)
CONTROL METHODS
* To determine concentration
WIPE TEST
