uconn biogrid reu summer 2008 primer design for multiplex pcr
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UConn BioGrid REU Summer 2008 Primer Design for Multiplex PCR. Nikoletta DiGirolamo. Primer Design: The Challenge. One criteria to achieve highly specific amplification product in MP-PCR reactions is to keep the concentration of amplification primers low. - PowerPoint PPT PresentationTRANSCRIPT
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UConn BioGrid REU Summer 2008
Primer Design for Multiplex PCR
Nikoletta DiGirolamo
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Primer Design: The Challenge
DPS-HDR MDPSP DPS-HDE MDPSP with Errors
NEW
New Algorithms for Problems
One criteria to achieve highly specific amplification product in MP-PCR reactions is to keep the concentration of amplification primers low.
In research, the dilemma associated with the primer minimization is formulated as the Multiple Degenerate Primer Selection Problem (MDPSP).
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Primer Design: What is PCR?
Primers
3' 5' 3' 5' 3' 5'
Denaturation Hybridization Elongation 2nd round
The Polymerase Chain Reaction
5' 3' 5' 3' 5' 3'
5' 3'
3' 5'
Targeted DNA
3' 5' 5' 3'
The Multiplex Polymerase Chain Reaction
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Primer Design: What is a Degenerate Primer?
A{C|G}TA{A|G|T}CA: ACTAACAACTAGCAACTATCAAGTAACAAGTAGCAAGTATCA
Degenerate Positions
Degeneracy = 6
Why use degenerate primers?
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Primer Design:The Problem
The Higher the value of degeneracy, the greater the primers'
concentration!
Aim : minimizing the number of degenerate primers
bounded degeneracy maximizing coverage (dmax)
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The Multiple Degenerate Primer Selection Problem (MDPSP):
Definition 1:
Find a set of degenerate primers with the length of l , degeneracy at most d, and maximum coverage that would collectively amplify all the n input sequences.
Primer Design:Definition 1
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Primer Design:Earlier Works
Algorithms:
HYDEN: Maximum Coverage DPD ( Linhart and Shamir [2002])MIPS: Multiple Degenerate Primer Design (Souvenir et. Al, [2007])DPS: Maximum Coverage DPDP (Balla et. al, [2007])
DPS-HD: Multiple Degenerate Primer Selection Problem (Balla et. al,[2007])
Common goal: maximize coverage at each step of the iteration
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Primer Design:Hamming Distancel-mers
Hamming Distance (HD): u: ACGTAACT v: ACTTACGT
HDuv = 3
# Introduction of DPS-HD
L-mer : a substringof sequence n with the length of l
(m – l + 1)
n
l-mers
Last l-mer
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Primer Design:Algorithm DPS-HDR1
s1
Sn
k
m
(m – l + 1)
u 1
n
(m – l + 1)
4 3 2 5
3 4 6 7
2 6 8 5
4 8 3 2
4 4 9 2
HDmin = 2
Step #1 Step #2
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Primer Design: Algorithm DPS-HDR 2
S1
kK
HD = 0
Step #3
Random v
Hdmin = 2
u
v
Degenerate primer : u'
vv
v
v
Coverage of u = 2
Sn
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Primer Design:Definition 2
Multiple Degenerate Primer Selection Problem
with Errors (MDPSPE)
Error ( E ): is an input constant corresponding to the number of mismatches allowed between primer u and the input sequence to be covered.
Definition 2 : given n DNA sequences, each with the length of m, and integers d, l, and E, find the set P of degenerate u that would match all the n input sequences with up to E errors (mismatches) such that u ∊ P has l length and at most D degeneracy.
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Primer Design: New AlgorithmsDPS-HDE
HD = 2HD = 4
HD = 0
HD = 5HD = 3
HD = 5
E = 3
HD <= 3
Coverage of u = 3
*Increases the coverage of the primers
*Decreases the cardinality of the final degenerate primer set
*Reduction in running time
S1
Sn
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Primer Design: Results: 1 - 2
D = 10000, l = 15 D = 100000, l = 15
DPS-HDR
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D = 100000, l = 15
D = 10000, l = 15
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Primer Design: Future Work
Optimize the efficiency of the Perl program
Implement the algorithm not only on random but also on real biological data
Initiate collaboration with molecular biologists to validate the algorithms performance in wet-lab experiments
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Thank You
July 31, 2008
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DPS-HD vs. DPS-HDRPrimer Design :
Set of vs
u
u'
u' with the best coverage
Step #3
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Primer Design: ComparisonMIPS, DPS, DPS-HD
D = 10000, l = 15
DPS-HD
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Primer Design: ComparisonMIPS, DPS, DPS-HD
D = 100000, l = 15
DPS-HD