Primer Design Flashcards
• A_______ is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing.
primer
are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish it to anneal.
primers
• Polarity/direction of primer:
5’ to 3’
2 TYPES OF PRIMER
Downstream = Reverse
Upstream = Forward
WHY ARE PRIMERS IMPORTANT?
Primers are what gives polymerase chain reaction its specificity.
Good primer design = PCR reaction will work great
CANNOT PROCEED TO EXTENSION IF PRIMER DOES NOT WORK
PRIMER DESIGN
• GOAL:
Make a specific and efficient primer
Bind to target DNA specifically (no unwanted binding)
Must be able to facilitate extension
The _____end of the primer should correspond completely to the template DNA strand so elongation can proceed
3’
The specificity of primer is often controlled by its____ (generally of 18-24 base pairs)
length
15-30bp
PRIMER LENGTH
Too short (<15 bp)
Too long (>30 bp)
non-specific amplification of PCR products
slower hybridization with the template DNA and inefficient annealing; require greater melting/annealing temperature
Denaturation Temperature
Annealing temperature
Extension temperature
90-96°C
55-60°C
70-72°C
ANNEALING TEMPERATURE
Vs MELTING TEMPERATURE
BINDING TO THE DNA TEMPLATE
PARTIALLY SEPARATING FROM THE
DNA TEMPLATE
Melting in_______ - hydrogen bonds between double stranded DNA
Melting in______ - half of the primer has annealed in the DNA template (partially separating from DNA template)
DENATURATION
ANNEALING
The temperature at which the primer hybridize with the template
DNA
Binding of the primer towards the DNA template.
ANNEALING TEMPERATURE
Ideal annealing temperature must be low enough to enable_____ and must be high enough to_____
hybridization between the primer and the template DNA
prevent mismatch hybrids from forming
If melting temp is 60
What is the optimal annealing temp?
55-57
The temperature at which half of the primer has annealed in the
DNA template.
MELTING TEMPERATURE
MELTING TEMPERATURE
Tm = (4x [G + C]) + (2 x [A + T]) °Celsius
The ideal primer has a melting temp/annealing temp in the range of_____
55-65C
PCR primers should maintain a reasonable GC content (should be between_____)
40-60%
Too much GC-
requires higher temp to anneal and
high temp could denature the target DNA template.
Too low GC
Primer can be unstable and could anneal other than your target template.
the presence of a guanine (G) or cytosine base in the last 5 bases (3’ end) of a PCR primer
GC CLAMP
GC CLAMP
the presence of a..
guanine (G) or cytosine base in the last 5 bases (3’ end) of a PCR primer
it is not recommended to include____ G or C in the last 5 bases.
more than 2
More than 2 GC in GC clamp may…
Reduce specificity
Increase temperature
Hairpin formation
If no GC found in GC clamp?
Unstable
Must have at least 2
the likelihood that the primer will bind to itself and to the other primer in the pair.
Self-complementarity
Self-complementarity score -should be…
less than 8 (<8)
Self 3’ complementarity
-should be______, so that primers will not bind to itself and into the other primer in the pair.
less than 3 (<3)
- If this state exists, a primer will fold back on itself and result in an unproductive priming event.
Hairpin
Hairpins
- If this state exists, a primer will…
fold back on itself and result in an unproductive priming event.
Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself or to the other primer used in PCR reactions
Primer dimerization
Example - One forward primer binds to the same forward primer or at reverse primer.
Primer dimer
How do we prevent self-complementarity?
Self-complementarity score should be less than 8 (<8).
Self 3’ complementarity should be less than 3 (<3), so that primers will not bind to itself and into the other primer in the pair. Predict Primer dimer
Self-complementarity score is useful to predict…
• Self 3’ complementarity predicts…
possible secondary structure of the primer (Hairpin).
Primer Dimer.
Self-complementary is more than 8…
This determines the possible outcome for hairpin appearance
More than 3 self 3’ complementary score…
Beyond 3 determines the possibility of having a primer dimer interaction.
