UConn BioGrid REU Summer 2008

Primer Design for Multiplex PCR

Nikoletta DiGirolamo

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).

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

Primer Design: What is a Degenerate Primer?

A{C|G}TA{A|G|T}CA: ACTAACAACTAGCAACTATCAAGTAACAAGTAGCAAGTATCA

Degenerate Positions

Degeneracy = 6

Why use degenerate primers?

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)

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

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

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

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

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

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.

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

Primer Design: Results: 1 - 2

D = 10000, l = 15 D = 100000, l = 15

DPS-HDR

D = 100000, l = 15

D = 10000, l = 15

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

Thank You

July 31, 2008

DPS-HD vs. DPS-HDRPrimer Design :

Set of vs

u

u'

u' with the best coverage

Step #3

Primer Design: ComparisonMIPS, DPS, DPS-HD

D = 10000, l = 15

DPS-HD

Primer Design: ComparisonMIPS, DPS, DPS-HD

D = 100000, l = 15

DPS-HD