Nucleotide Sequence Analysis

Week9
Lecture: Polymerase Chain Reaction
Lab: NET Primer & NCBI-Primer BLAST
 Polymerase Chain Reaction
Developed in the 1980s by Kary Mullis
Technique for making copies, or amplifying, a specific
sequence of DNA in a short period of time
In vitro replication of DNA
Needs/Substrates:
Template
Unzipping of Helix (?)
Primers
Primer Annealing (?)
DNA Polymerase
dNTPs
Zipping of Helices (?)
First PCRs- Rudimentary with different temperature baths
Thermocycler: instrument that can go through a series of temperature
changes and can cycle through them
DNA polymerase
Taq DNA polymerase isolated from a species known as Thermus aquaticus that thrives
in hot springs
 PCR Cycle
Each cycle consists of three stages
Denaturation (melting -95C, 2min))
Annealing (hybridization- 60-65C, 30s)
Extension (elongation- 68-72C,
1min/kb)
At the end of one cycle, the amount of
DNA has doubled
Cycles are repeated 30-35 times
Number of copies of DNA obtained
after 'n' cycles = 2(n+1)
Advantage of PCR
Ability to amplify millions of copies of
target DNA from a very small amount of
starting material in a short period of
time
Applications
Cloning
Forensic Science
Genome Sequencing
Clinical Diagnosis
Primer design: most important step of PCR
Two main concerns with PCR primers
Mis-priming: Non specific Products
No product
Primers should be about 20 nucleotides long
What happens if primers are too short? too long?
Primer Tm around 63-65C is ideal
Tm dictates Ta (annealing temperature)
Tm below about 55 undesirable: why?
Primers Tm should be within 1-2C of each other why?
Primer Design: Secondary Structures
Hairpin loop
formed when primer folds back upon itself
held in place by intramolecular bonding
Not desirable- why?

The primer sequence is ATCGATATTCGAAGAT

It forms two hairpins:
3' end hairpin where the primer folds back upon itself and first and last 3
bases bond together
internal hairpin where 2nd-5th and 9th-12th bases bond together
 Primer Design- Primer Dimers
Complementarity between primers: Bonding of two primers.
Primer should not be self-complementary.
Not desirable- why?
Cross & Self Dimers
Cross Dimer (example)

Sense primer sequence is ATCAGCTGTAGAT

Anti-sense primer sequence is ATAGTGTAGAT
Forms one cross dimer at the 3' end
 Self Dimer (example)

The primer sequence is ATCAGCTGTAGAT

It forms 2 dimers:
internal dimer where 3rd-8th bases of primer in 53' (starting from
5') bond with 6th-11th bases (starting from 3') when primer is placed in
reverse direction
3' end dimer where the last 3 bases (starting from 5') of primer
placed in 53' direction bond with last three base (starting from 3')
placed in reverse direction.
 Primer design: Stability of 5 & 3 end; GC Clamp
Desirable: Stable 5 end- makes the primer more sticky and increase specificity.

Desirable: Unstable 3 end- less sticky primer at 3 end- why?

GC clamp: short stretch of G/Cs (about 3-4) at 5 end of the primer GC

hybridization is stronger (why?) and this makes the primer stick well at the 5
end- increases stability of 5 end.

The GC clamp at 3' end used to be old school thinking but more analysis have
shown that if you have anything more than 2G/C within last 5 bases of the primer-
you lose specificity. So it is no longer recommended.

Can have GC clamp in the middle of the primer.

One can argue that why presence of GC at 5' end does not lead to false priming
and loss of specificity- exact reasons are not known- it is an observation
researchers have made. The reason speculated is that because 3' end is what gets
extended and hence most responsible for non-specific PCR products.

Net primer reports 5 and 3 end stability as negative Gs- so be careful while
making your predictions today.