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