Design and Applications in Molecular Biology Research: Primer Design Module 11 http://www.csd.uwo.ca/courses/CS461b/

Acknowledgment Jennifer M. Anderson, Ph.D. Li Liu, M.D. Interdisciplinary Center for Biotechnology Research, University of Florida, June 10, 2003 Worachart Sirawaraporn, Dept of Biochemistry, Mahidol University, January 28, 2005 Zhang Univ of Western Ontario, CS 461b/661b: Bioinformatics Tools and Applications http://www.csd.uwo.ca/courses/CS461b/ on Jan 20, 2005

General knowledge of DNA replication

PCR

Considerations About Primers Characteristics of primers: Thoughts on primer design: Uniqueness Specificity Specific for the intended target sequence (avoid nonspecific hybridization) Length Base Composition Internal Stability Stability Form stable duplex with template under PCR conditions Melting Temperature Annealing Temperature Internal Structure Compatibility Primers used as a pair shall work under the same PCR condition Primer Pair Matching

Where do primers come from ? Chemically synthesized as ssDNA Typically 15-25 nucleotides in length The nucleotide sequence determined by - prior knowledge of target DNA sequence - reverse translation of protein sequence

Primer design elements Primer length GC% Annealing 3’ complementary between primers G&C runs at the 3’ end Palindrome sequences

Good Primer’s Characteristic A melting temperature (Tm) in the range of 52°C to 65°C Absence of primer dimerization capability Absence of significant hairpin formation (>3 bp) Lack of secondary priming sites Low specific binding at the 3' end (ie. lower GC content to avoid mispriming)

Uniqueness NOT UNIQUE! UNIQUE! There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA. There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome) Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’ TGCTAAGTTG CAGTCAACTGCTAC TGCT AGTTG A Primer candidate 1 5’-TGCTAAGTTG-3’ NOT UNIQUE! Primer candidate 2 5’-CAGTCAACTGCTAC-3’ UNIQUE!

Length Primer length has effects on uniqueness and melting/annealing temperature. Longer the primer = more unique Longer the primer = higher melting/annealing temperature. Generally speaking, the length of primer has to be at least 15 bases to ensure uniqueness. Usually, we pick primers of 17-28 bases long. This range varies based on if you can find unique primers with appropriate annealing temperature within this range.

Base Composition Base composition affects hybridization specificity, melting/annealing temperature and internal stability. Random base composition is preferred. We shall avoid long (A+T) and (G+C) rich region if possible. Template DNA 5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’ TGCCCGATCATGCT GCCCG CAT T T AT GC Usually, average (G+C) content around 50-60% will give us the right melting/annealing temperature for ordinary PCR reactions, and will give appropriate hybridization stability. However, melting/annealing temperature and hybridization stability are affected by other factors, which we’ll discuss later. Therefore, (G+C) content is allowed to change.

Melting Temperature Melting Temperature, Tm – the temperature at which half the DNA strands are single stranded and half are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNA has a higher Tm due to more H bonds. Calculation (Base Composition): Tm = 59.9 + 0.41*(%GC) - 600/length Other more accurate methods are available.

Primer Annealing Temperature Simple Method (Thein and Wallace 1986): A and T = 2o C G and C = 4o C So, add up the number of A and T in the primer and multiply by 2 and then add up the number of G and C and multiply by 4. Add these two number together to get the approximate annealing temperature. Exercise: If the following was your primer set (below) using the method above, what would be the annealing temperature you would use for the reaction: Forward ATG TGT GAC GAT GAG GTT GC Reverse TGG CTG GGG CAT TGA AGG TC

Stringency in Primer Annealing Stringency determines the specificity of the amplified DNA product. Tanneal is the most significant factor affecting the stringency in primer annealing. Tanneal : too low  less stringent  primer matches elsewhere too high  more stringent  primer may fail to match Other factors: GC%: GC pairs are more stringent than AT paris Salt & Buffer

Internal Structure If primers can anneal to themselves, or anneal to each other rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided. However, sometimes these 2 structures are harmless when the annealing temperature does not allow them to take form. For example, some dimers or hairpins form at 30 C while during PCR cycle, the lowest temperature only drops to 60 C.

Primer Pair Matching Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them. One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3 C. The closer their Tanneal are, the better.

Summary ~ when is a “primer” a primer? 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’

Summary ~ Primer Design Criteria Uniqueness: ensure correct priming site; Length: 17-28 bases.This range varies; Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible; Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming. Melting Tm between 55-80 C are preferred; Assure that primers at a set have annealing Tm within 2 – 3 C of each other. Minimize internal secondary structure: hairpins and dimmers shall be avoided.

Diagram for PCR Primer Design Sequence from which to choose primers Primer Design Results of search, including suggested annealing temperatures shown in list PCR reaction parameters Primer Selection Rules Primer design is an art when done by human beings, and a far better done by machines. 

Web sites related to primer design PCR primer designer: Primer3 Single sequence input: http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi Others: Oligo, BioTools, GCG, GeneFisher, Primer!, Web Primer, NBI oligo program, etc Lists of other PCR software Input protein alignment: http://blocks.fhcrc.org/blocks/codehop.html Input nucleotide sequences or nucleotide alignment: http://bibiserv.techfak.uni-bielefeld.de/genefisher/ List of primer design sites: http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html Melting temperature calculation software: BioMath: http://www.promega.com/biomath/calc11.htm

Common problems in primer design self complementarity upper-lower primer complementarity excessive thermal stability computer aided primer design

Hairpin formation

Primer dimer formation

Internal stability of the primers Primers with stable 5’ termini give the best performance: reduces false priming on unknown targets Low 3’ stability prevents formation of duplexes that may initiate DNA synthesis: 5’ end must also pair in order to form a stable duplex Optimal terminal ∆G ~ 8.5 kcal/mol; excessive low ∆G reduces priming efficiency

Common problems in PCR Pre-PCR mispriming Non-template nucleotide addition “Plus A artifacts”

Pre-PCR mispriming undesired annealing between primers themselves or with ssDNA molecules at low temperature DNA polymerase extends the misannealed primers, adding nucleotides to their 3’ end, rendering these modified primers unable to anneal to their intended sites and resulting in amplification of unwanted DNA

Solving Pre-PCR mispriming Performing “HOT START PCR” Reaction mix minus Taq Template Primers dNTPs Transfer to thermocycler 100 oC denaturation 80 oC hold Add Taq polymerase Start thermocycler

Degenerate Primers Used to isolate novel genes on the basis of similarity and/or amino acid sequences To amplify (fish out) conserved sequences of a gene or genes from the genome of an organism. Used when attempting to design “universal” primers or when no unique area is found among aligned sequences

Nucleotide Represented Degenerate Primers Abbreviation Letter Nucleotide Represented A  A C  C G  G T  T R  AG Y  CT M  AC K  GT W  AT S  CG B  CGT D  AGT H  ACT V  ACG N  ACGT eg: 5’-TCG AAT TCI CCY AAY TGR CCN T-3’ Y = pYrimidines = C / T (degeneracy = 2X) R = puRines = A / G (degeneracy = 2X) I  = Inosine  = C / G / A / T  N = Nucleotide = C / G / A / T (degeneracy = 4X)  

Degenerate Primers A potential degenerate primer: ATG A[T/C]A AA[T/C] ATC CGA AAA AC[T/A/C]C A H Y Y

Degenerate Primers Use greater primer concentration (>50 pmol) to compensate for degeneracy. Competitive inhibition can be a problem Multiple site annealing No products formed

Universal Primers Primers can also be designed to amplify multiple products. We call such primers “universal primers”. For example, design primers to amplify all HPV genes. Strategy: Align groups of sequences you want to amplify. Find the most conservative regions at 5’ end and at 3’ end. Design forward primer at the 5’ conservative region. Design reverse primer at the 3’ conservative regions. Matching forward and reverse primers to find the best pair. Ensure uniqueness in all template sequences. Ensure uniqueness in possible contaminant sources.

Semi-Universal Primers Primers can be designed to amplify only a subset of template sequences from a large group of similar sequences. For example, design primer to amplify HPV type 1 and type 6 gene, but not other types. Strategy: Align all types of HPV genes. Identify a subset of genes that are more similar to each other than to other subsets. In this case, type 1 and type 6. Find the 5’ and 3’ regions that are conserved between type 1 and type 6, but are variable in other types. Design forward primers from the 5’ region and reverse primers from the 3’ region. Matching forward and reverse primers to find the best pair. Ensure uniqueness in all template sequences. Ensure uniqueness in possible contaminant sources.

Multiplex PCR Multiple primer pairs can be added in the same tube to do the PCR Good for amplifying multiple sites Application example: genome identification Design difficulty Melting temperatures should be similar No dimer formulation

Primer Design – Example Design a pair of primers for sequence “NM_203378” in NCBI GenBank, so that the coding sequence of human myoglobin will be amplified using PCR reaction. Between 156..620

Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

Primer3

Primer3