amplification techniques Flashcards
Hybridization
The annealing (pairing) of two nucleic acid strands
Target
The specific gene or nucleic acid sequence that is of interest
Primer
- An oligonucleotide that serves to initiate polymerase-catalyzed addition of nucleotides by annealing to a template strand
- A short stranded nucleic acid that acts as a starting point for nucleic acid synthesis
Probe
- A nucleic acid which is used to identify a target sequence by hybridization
- A small nucleic acid that is used to detect the presence of a specific target sequence
- Usually labelled
Label
• Any substance with a measurable property that can be attached to an antigen, antibody or binding substance
Amplification Methods ( 3 )
Techniques that increase:
• The amount of the nucleic acid target (Target Amplification)
• The detection signal (Signal Amplification)
• The probe (Probe Amplification)
In practice, amplification techniques may achieve more than a million-fold amplification in less than an hour!
Target amplification
increasing the amount of target nucleic acid
The sequence of interest is copied many times by in vitro methods
Areas outside the target are not amplified
Signal amplification
increases the signal
amount of target stays the same but the signal is increased
Probe amplification
probe is amplified only in the presence of the target
Polymerase Chain Reaction (PCR) purpose
target amplification technique
Most common molecular technique employed in lab medicine
Method for amplifying a small, defined fragment of DNA from a complex pool of nucleic acid
• Pick one piece of DNA that is of interest, and amplify it millions of times
amplifies low levels or specific DNA sequences from a sample which can then be used for follow up testing
Sometimes PCR is used to determine IF something is present, and sometimes it is used as a starting point to generate more
DNA to test
Polymerase Chain Reaction (PCR) required materials
• A thermostable DNA polymerase
• Deoxynucleotides of each base (collectively referred to as dNTPs)
• The target sequence
• A pair of oligonucleotides –referred to as primers
- Complementary to opposite strands flanking the sequences you
want to amplify
• Magnesium
- Required for the proper function of Taq polymerase
• Buffer
- Standard buffers are composed of Tris-HCl and a salt like
KCl at a pH of 8.3
• PCR requires an instrument known as a thermocycler which will
take the samples through multiple steps of changing temperatures
what is another name for PCR product
Amplicon
PCR steps
- Denaturation
target duplexes are denatured into single strands by heat
95 degrees - Annealing
As the mixture is cooled, primers provided in great excess specifically anneal to complementary sequences on the target - Extension
Once the primers are annealed, the action of the polymerase synthesizes two additional DNA strands containing the primers as the 5’ ends
- The primers are placed close enough together so that the polymerase extends each strand far enough to include the priming site of the other primer
70 degrees- Optimal temperature for Taq polymerase to create
new complementary DNA
4.Repeat
The second cycle also begins with denaturation, but now there are twice as many strands available for primer annealing and subsequent extension
- continues ~35 cycles or until plateau is reached
3 temperatures for PCR and reasoning
• High temperature to denature the target sequence
~95
• Low temperature that allows annealing of the primers to the target
~50
• Intermediate temperature that is optimum for polymerase extension
~70 optimal for taq polymerase
PCR efficiency depends on
- Concentration of primer and polymerase
- Temperature-cycling protocol
- The presence or absence of polymerase inhibitors
- If the efficiency of each cycle is optimal (100%), the number of target sequences will double with each cycle
- Amplified products accumulate exponentially in the beginning cycles of PCR however the efficiency falls, and the amount of product will plateau
using 0.5 μL of each primer, the maximum DNA
concentration achievable is about 1011 copies/μL
reasons PCR plateaus
• Exhaustion of components
- Primers
- Nucleotides
• Competition between primer and product annealing as the single
strands of product are at such high concentrations that they anneal to
each other rather than primers
PCR Product Detection
Simple Gel Electrophoresis
• Involves fluorescently labeling one of the primers so that after PCR, the fragments are able to be sized on a DNA sequencing device
• Can be used in combination with ethidium bromide staining
Real-time PCR
• Adds a fluorescent dye or probe before amplification
• Allows thermocyclers equipped with optical detection to analyze the reaction either
• As it progresses
• After the reaction is complete
PCR Primer Design
-primers determine the specificity of the reaction
• ssDNA Designed to contain sequences that are complementary to sites flanking the region of interest (the target sequence)
• Primers should be designed so that the forward and reverse primers have the
same annealing temperature for optimal hybridization
Primer Dimer
An artifact which is often observed in PCR
• Result when forward and reverse primers bind with each other, insteadof the target sequence, therefore amplifying the primers
PCR contamination prevention
A small amount of contamination in a sample can easily cause a false-positive result
Contamination can be found in/on: • Reagents • Pipettes / Glassware • Work surface • Gloves
** If possible, the most effective way to avoid contamination is to contain the product in a closed tube and not let any escape
Precautions to minimize contamination include the use of:
• Physically separated areas for preamplification and post amplification steps
• Positive-displacement pipettes to minimize aerosol contamination
• Pre-aliquoted reagents
PCR No template control
“a Blank”
- This uses the same master mix, primers and nucleotides as the samples, but substitutes DNA-free water instead of DNA.
- The no template control should NOT show amplification – if amplification is seen, it indicates contamination
PCR Negative control
used to test that the primers are specific for the desired target.
- This uses the same master mix, primers and nucleotides as the samples but uses a DNA sample that is known to be negative for the target sequence
- If the negative control shows amplification – it indicates either contamination or non-specific/ poor primer design
PCR Controls: Inhibition Control (False Negative)
- Clinical samples contain unpredictable amounts of impurities which may inhibit polymerase activity
- If polymerase activity is inhibited, NO amplification will be seen, regardless of the presence of the target nucleic acid – this could lead to false negative results
• Typically, to rule out the presence of an inhibitor, a control nucleic acid sequence is added to the sample
- Failure to amplify this control indicates that the sample needs to be purified to remove inhibitors of amplification
2 types of modified PCR
asymmetric PCR
Allele - specific PCR
Asymmetric PCR
Uses different concentrations of the two primers
- generate more of one strand than the other
• Resulting in single-stranded excess of one strand
• The single-stranded product is then able to be directly
hybridized to probes without denaturation
Allele- specific PCR
preferential amplification of one genetic allele over another
• The 3’ end of one primer is placed at the polymorphic site and is extended only if it is completely complementary to the
target
• This is useful for distinguishing a gene from its pseudogenes and for genotyping SNVs
DIgital PCR
Also known as single-molecule PCR
• Limits the amount of template that is distributed across
reaction compartments
• Some compartments will have no template
compartments may be: • Very small droplets in a water/oil emulsion • Emulsion PCR • PCR colonies (polonies) within a gel • On the surface of a flow cell • On Microbeads
thousands of reactions are analyzed, very exact quantification can be obtained
EX. copy number changes and rare mutations in a population of tumor cells are quantified
- Can assess whether multiple variants are on the same or different chromosomes
- This is the first step for many high-throughput sequencing methods
reverse transcriptase- PCR
The most sensitive technique available for detecting and quantifying
mRNA
• Able to evaluate the transcript from a single cell
• Uses reverse transcriptase to synthesize a complementary strand of
DNA (cDNA) from an RNA template
• The resulting cDNA is then used as the template in the PCR assay
Can be one in one step or two
• One-step uses a single tube to conduct the reverse transcriptase
step as well as the PCR
- Minimizes the potential carryover of amplicons to the working
environment & minimizes pipetting errors
• Two-step is useful when more than one type of transcript needs
to be detected
• cDNA from one transcript can be removed and added to a
second tube with primers specific for one sequence (can set up multiple Rxns)
RT-PCR is used to detect RNA viruses
can quantify amount of viruses present
- used to monitor viral load for HIV& Hepatitis patients
also used for quantitative analysis of gene expression, detection of
human genes involved in disease and detection of cancers
Quality Control for Reverse Transcription PCR
Genomic DNA in the specimen may contaminate the RNA template so it should be treated with a DNase
• An enzyme that hydrolyzer DNA BEFORE the RT-PCR begins
Endogenous Control
• A gene whose expression level should not differ between samples
• Sometimes called a reference control or internal control
• It controls for variation in RNA content, RT efficiency, RNA integrity
and overall sample handling
- Often β-actin, glyceraldehyde phosphate dehydrogenase (GAPDH), 18S rRNA
- The endogenous control MUST be positive for the assay to be valid
Target Amplification Techniques
Targets either DNA or RNA but generates RNA as its amplified
product
amplified without temperature cycling
• Requires a reverse transcriptase – RNAse H
• Requires RNA polymerase
The method may be applied to single strand RNA or dsDNA
• An initial heat denaturation is needed if a dsDNA template is
used
useful if the target is a viral RNA from patients carrying HIV or
Hepatitis C in blood bank nucleic acid testing
• This method is currently used to detect some microorganisms, like
Mycobacterium tuberculosis, C. trachomatis and N. gonorrhoeae
Ligase Chain Reaction (LCR
probe-based DNA amplification technique that uses four probes
• Uses a DNA polymerase and DNA ligase
Oligonucleotide pairs hybridize to target sequences
• The bound nucleotides are separated by a small gap at the target
site
• DNA polymerase uses nucleotides in the reaction mixture to fill
this gap, creating a ligatable junction
• Once this gap is filled, DNA ligase joins the oligonucleotide
pairs
Cycles of denaturation and ligation lead to the accumulation of
amplification products
uses both Target and Signal Amplification techniques
Signal Amplification Techniques
Branched-Chain Signal Amplification
• Creates branched DNA (bDNA)
• Hybridizes the target nucleic acid to multiple capture probes which are affixed to a microtiter well
Followed by hybridization to a series of:
• Extender probes
• Preamplifier probes
• Amplifier probes
• The final highly branched amplifier probe has multiple copies
of signal generating enzymes that act on a chemiluminescent
substrate to produce light
Probe Amplification Techniques
Rolling Circle Amplification (RCA)
• amplify DNA from very small amounts of starting material
- In the presence of a template, a linear probe is ligated to form a circular template
- This anneals to the primer
- The circle is replicated continuously by a polymerase and one or more primers
- The amplified probe can then be detected and indicates the presence of the target
Quantification after Amplification
usually requires calibration with known amounts of target or a target mimic
• It is accomplished by comparison to an internal standard that can be
added at the time of sample processing
• Internal standards can include
• DNA fragments
• Plasmids
• RNA packaged into synthetic phage (virus) particles
• Both the sample and the internal standard should be affected
identically by any variation in reaction activity
• Real-time PCR is a simpler and more powerful approach to
quantification and will be discussed later
generic and specificdetection methods for nucleic acids
Generic techniques measure the total amount of nucleicacid
•Ultraviolet Spectrophotometry
•Fluorometry and Fluorescent Staining
Specific techniques measure a particular sequence
•Sequence Specific Labels
