CHRISTINE P.

ALAYON  3'terminal sequence

CELL AND MOLECULAR BIOLOGY (SY 17-18)
POLYTECHNIC UNIVERSITY OF THE
PHILIPPINES
Polymerase Chain Reaction
PCR is a method of exponential DNA amplification
 Sensitive, reliable, rapid,convenient and
efficient
 Nobel: Kary Mullis 1993
 Small amount of DNA: millions of copies in
hours
Basic Principles of PCR
1. Strands of template DNA (or RNA) are
separated melting (denaturation)
2. Forward Primer binds to one strand of
template, Reverse Primer to other strand (annealing)
3. DNA polymerase extends 3’ end of each
primer, copying template (extension)
4. New strands are separated by raising
temperature, allowing both original DNA and copies
to act as templates
5. Repeat steps 2-4 many times (profile)
PCR AMPLIFIES DNA (AND RNA AT TIMES)
Step1: Denaturation
Step 2: Annealing
Step 3: Elongation/Extension
o strong bonding base (G or C) at end
o no runs (3 or more) of G or C at
end
o avoid poly T sequences
LENGTH
Length affects the melting point (Tm) during
annealing:
temp at which ½ of the DNA duplex will dissociate
to become single stranded
-> duplex stability
Too short: (less than 15)
-> less specific (A), low Tm
Too long: (more than 25)
-> less amplification (B)due to high Tm Dimerization
(C).
Base Composition
- GC increases the Tm so has to be limited to 40-60%
Melting Temperature
Tm: temp at which ½ of DNA is SS.
Too low: Tm <50: nonspecific annealing
Too high: Tm> 65: secondary annealing
Normally tested for PCR: (+5 and -5 from primer
Tm)
Tm(K)={ΔH/ ΔS + R ln(C)}, Or Melting
Temperature Tm(oC) = {ΔH/ ΔS + R ln(C)} -
273.15 where ΔH (kcal/mole) : H is the Enthalpy, S
is the entropy of the system.

3’ Terminal Sequence
(GC clamp): G or C bases within the last 5 bases
from the 3'end of primers -> helps promote specific
binding at the 3' -> stronger bonding of G and C
bases.
* More than 3 G's or C's should be avoided: dimers
again!

Basic Steps in Designing a Primer

1. Get sequences from an information bank
2. Align sequences using an algorithm
WHAT IS A GOOD PRIMER? 3. Generate a consensus sequence
Primers 4. Use sequence (3) as target for primer design
Success is dependent on a good primer 5. Screen primer candidates
A primer attaches during annealing. Initially it has to 6. BLAST primers to check for specificity
be 3 things:
 Short: quick to attach (single stranded) PRIMER CRITERIA:
 Sensitive: matches template Length of product: 300-1500 bp (500 optimum)
 Specific: Amplifies one target ONLY (as Pair GC composition: (45-55%)
much as possible) Melting temperature difference: (not > 5 C)
Initial Criteria Self complimentarily: lowest possible for pair

 Length (18-25 bases)

 Base composition (45-55% GC)
 Melting temperature (55-80 C)