Genome Scale PCR
Infidelity Search
(The Promiscuous Primer Problem)
Goal: An efficient search for the
presence of potential undesired PCR
products that scans through 3 billion
bases of human DNA.
Conditions for Mispriming
►
1. First 3 bases on 3’ end of
primer must perfectly
complement 3 bases in
sequence.
►
2. Melting temperature (Tm) of
the potential duplex > x
(annealing temperature of PCR
reaction – y).
►
3. The position of the
mismatch(es) between the
primer and genomic DNA will
affect the binding of the
polymerase, so must account for
differential binding based on the
position of the mismatch(es)
(Real-Time PCR studies will
address this).
►
4. Forward and reverse primers
must be within z bases of each
other.
Central Issues of Infidelity Search
1. Size of human genome: ~ 3 billion base pairs.
 Run Blast search locally or presort genome as
prefix tree and write search program.
2. Tm Calculations are computationally expensive
to repeat billions of times.
 Precalculate all sequences that will misprime
rather than calculate Tms “on the fly.”
The Algorithm
Tm 
DH 
DS   R ln CT

DTm  Tm1 
DH  dHT  dHT '
DS  K  dST  dST '
We know the primer Tm, DH, and DS. Scan all possible single mutations,
and calculate their Tms going from most to least stable. Repeat process
for two mutations, etc. Once we hit a threshold value, we know the
primer will not bind with anything else.
The Search
After calculating all the possible sequences that will misprime with our primer,
we can then do a string search.
The ideal answer:
1. Run a batch Blast locally with all the possible sequences.
2. If a forward hit and reverse hit are < x bases apart, we say that is a
misprime!
The hard answer:
1. Write a search program of the presorted genome and hold misprime indices
to compare forward and reverse primers; then check to see if they exist < x
bases apart.
A
AA
AAA
AAC
AC
AAG
AG
AAT
AT