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UConn BioGrid REU Summer 2008 Primer Design for Multiplex PCR

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.

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UConn BioGrid REU Summer 2008 Primer Design for Multiplex PCR

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  1. UConn BioGrid REU Summer 2008 Primer Design for Multiplex PCR Nikoletta DiGirolamo

  2. 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. In research, the dilemma associated with the primer minimization is formulated as the Multiple Degenerate Primer Selection Problem (MDPSP). New Algorithms for Problems DPS-HDR MDPSP DPS-HDE MDPSP with Errors NEW

  3. Primer Design: What is PCR? The Polymerase Chain Reaction 5' 3' 5' 3' 5' 3' 5' 3' 3' 5' Primers 5' 3' 3' 5' Targeted DNA 3' 5' 3' 5' 3' 5' Denaturation Hybridization Elongation 2nd round The Multiplex Polymerase Chain Reaction

  4. Primer Design: What is a Degenerate Primer? A{C|G}TA{A|G|T}CA: ACTAACA ACTAGCA ACTATCA AGTAACA AGTAGCA AGTATCA Degenerate Positions Degeneracy = 6 Why use degenerate primers?

  5. Primer Design: The Problem TheHigherthe value of degeneracy, the greater the primers' concentration! Aim : minimizing the number of degenerate primers bounded degeneracy maximizing coverage (dmax)‏

  6. Primer Design: Definition 1 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.

  7. 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

  8. Primer Design: Hamming Distance l-mers Hamming Distance (HD): u:ACGTAACT v: ACTTACGT HDuv = 3 L-mer : a substring of sequence n with the length of l (m – l + 1)‏ n # Introduction of DPS-HD l-mers Last l-mer

  9. Primer Design: Algorithm DPS-HDR 1 Step #1 Step #2 (m – l + 1)‏ m u 1 s1 4 3 2 5 3 4 6 7 2 6 8 5 4 8 3 2 4 4 9 2 k (m – l + 1)‏ n Sn HDmin = 2

  10. Primer Design: Algorithm DPS-HDR2 Step #3 Random v Coverage of u = 2 S1 v u v k K HD = 0 v v v Hdmin = 2 Sn Degenerate primer : u'

  11. 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.

  12. Primer Design: New Algorithms DPS-HDE Coverage of u = 3 E = 3 S1 HD = 2 HD = 4 HD = 0 HD = 5 HD = 3 HD = 5 *Increases the coverage of the primers *Decreases the cardinality of the final degenerate primer set *Reduction in running time Sn HD <= 3

  13. Primer Design: Results: 1 - 2 DPS-HDR D = 10000, l = 15 D = 100000, l = 15

  14. D = 10000, l = 15 D = 100000, l = 15

  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

  16. Thank You July 31, 2008

  17. DPS-HD vs. DPS-HDR Primer Design : Step #3 Set of vs u' u u' with the best coverage

  18. Primer Design:Comparison MIPS, DPS, DPS-HD D = 10000, l = 15 DPS-HD

  19. Primer Design: Comparison MIPS, DPS, DPS-HD D = 100000, l = 15 DPS-HD

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