DIY Primer Design Oligonucleotides for Special Applications in Molecular Biology Alberto Catalano Kanematsu Labs, Institute of Haematology RPAH alberto.catalano@email.cs.nsw.gov.au Ph: 9515 7453 http://users.bigpond.net.au/albert/primers.htm Outline DNA Refresher: “DNA 101” PCR Introduction Oligonucleotide Primers for PCR Properties Specificity Self complementarity & primer-primer interactions General rules Primer Design Computer programmes: on internet & on PC Special PCR Applications DNA 101 The “Basics” B-DNA 2nm • Common form of DNA • Formed under high humidity conditions • Right-handed double helix • Major groove & minor groove • Sugar-phosphate backbones 3.4nm 10 nucleotides per turn Ramaswamy H. Sarma 1996 Sugar + Phosphate + Base Sugar + Phosphate form the backbone Base-pairing & DNA Stability 4 nucleotide bases in DNA Cytosine (C) pairs with Guanine (G) 3 hydrogen bonds Strong-pairing Adenine (A) pairs with Thymine ( T ) 2 hydrogen bonds Weak-pairing Stacking forces Van der Waals forces Influenced by nearest neighbour sequence Base-pairing Purines Pyrimidines Oligonucleotides Short single-stranded DNA Uses for oligonucleotides PCR, etc. Primer pairs Primer sets in multiplex assays Probes Sequence identification Gel shift assays Gene technology Synthetic genes Site-directed mutagenesis Oligonucleotide Choice Sequence Specificity GC content Target sequence location Avoiding repeat sequences Melting temperature Avoid Secondary structures Specificity Approximation of complexity (for a random sequence) 1 base = 41 ; 2 bases = 42 ; 3 bases = 43 ; ... n bases = 4n Biological sequences are not random! Check the oligos with BLAST Need to avoid complementarity with repetitive sequences in specific organism e.g. human Alu sequences, simple repeats Unwanted Self & Primer-Primer Interactions Primer self-complementarity At 3’-end can result in primer-dimer formation ||||||||||||||||||||||| ||||||||||||||||||||||||||| ||||||||||||||||||||||| ||||||||||||||||||||||||||| Internal homology : stem & loop structures Forward & reverse primer complementarity Primer-dimer formation between different primers ||||||||||||||||||||||| ||||||||||||||||||||||||||| ||||||||||||||||||||||| ||||||||||||||||||||||||||| Primer Length vs Purity Most oligonucleotide synthesis reactions are only 98% efficient. Each time a base is added, only 98% of the oligos will receive the base. As length increases, so does the probability that a primer will be missing a base Critical in mutagenesis or cloning reactions. Purification by HPLC or PAGE is recommended in some cases. Primer Length vs Purity Oligonucleotide length Percent with correct sequence 10 bases (0.98)10 = 81.7% 20 bases (0.98)20 = 66.7% 30 bases (0.98)30 = 54.6% 40 bases (0.98)40 = 44.6% Melting Temperature Oligonucleotide Factors: Primer length GC content i.e. Overall Sequence Sequence order due to stacking forces: nearest neighbour analysis Reaction Conditions: Salt concentration Primer concentration Presence of additives in reaction; e.g. formamide, DMSO, betaine, glycerol Relative absorbance at 260nm Hyperchromic shift & Tm 1.5 ssDNA 1.4 1.3 50% denatured 1.2 1.1 dsDNA 1 40 50 Melting temperature 60 70 80 Temperature (°C) Experimental determination of DNA melting temperature 90 Relative absorbance at 260nm Melting Temperature vs Annealing Temperature 1.5 ssDNA 1.4 1.3 Annealing temperatures 1.2 1.1 dsDNA 1 40 50 Melting temperature 60 70 Temperature (°C) 80 90 Mismatched Bases Mismatched bases Bonding between neighbouring bases is weakened by the mismatch. Therefore, the melting temperature is lowered General Rules for PCR Primers Innis & Gelfand 1990 1. 2. 3. 4. 5. 6. 7. Length : 17-28 bases G+C content : 50-60% GC clamp: terminal G, C, GC or CG Primer Tm : 55° - 80°C Avoid 3’-complementarity Avoid internal self-complementarity Avoid runs of 3 or more Gs or Cs near ends Steps of PCR DENATURATION PRIMER ANNEALING PRIMER EXTENSION BY POLYMERASE 20 to 21 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 21 to 22 |||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 22 to 23 DNA Denaturation 5’ 3’ ssDNA Complementary strands 3’ 5’ Primer annealing Annealing & Primer extension Primer Polymerase Template Primer Design Using computers to get primers that will work well Why use computers? Comparison of candidate primer sequence with repeat sequences from the species of interest Nearest-neighbour Tm calculations Primer self-complementarity analysis Identification of potential primer-dimer formation Analysis of large numbers of forward and reverse primer combinations to find a pair that fit the desired criteria for target sequence, product size, primer Tm, etc. PC based software Commercial packages: iOligo DNA Star PCR Help! (free demo) Oligo (free demo) Primer Premier (free demo) Free software: PerlPrimer Oligos GeneTool Lite (no longer supported) Terminology Forward primer Target: sequence to be included between primers Template: (genomic DNA or cDNA) Reverse primer Amplicon: resulting PCR product ANGIS Biomanager GCG Prime Basic selection of oligonucleotide primers for PCR and sequencing CodeHop designs a pool of primers containing all possible 11- or 12-mers for the 3' degenerate core region and having the most probable nucleotide predicted for each position in the 5' non-degenerate clamp region Primer3 “Primer3” http://frodo.wi.mit.edu/ Primer3 picks primers for PCR reactions, according to the conditions specified by the user. Primer3 considers things like melting temperature concentrations of various solutions in PCR reactions primer bending and folding Can also pick probes according to specified parameters Variants: e.g. PrimerQuest, with graphic output http://scitools.idtdna.com/Primerquest/ “Exon Primer” http://ihg.gsf.de/ihg/ExonPrimer.html helps to design intronic primers for the PCR amplification of exons needs a cDNA and the corresponding genomic sequence as input can avoid primers to be positioned across SNPs, using genomic sequence where SNPs are masked by N’s in input genomic sequence “Exon Primer” SNPs Multiple Targets: gene exons Template: genomic DNA Single amplicon for small exons/introns Multiple Amplicons Overlapping amplicons for large exons “CODEHOP” http://blocks.fhcrc.org/blocks/codehop.html COnsensus-DEgenerate Hybrid Oligonucleotide Primers PCR primers designed from protein multiple sequence alignments Amino acid alignments must be in Blocks Database format Intended for cases where the protein sequences are distant from each other and degenerate primers are needed “POLAND” http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html Calculates the thermal denaturation profile of double-stranded RNA, DNA or RNA/DNAhybrids based on sequence input and parameter settings e.g. Sequence: 70 44r CGCCAGCTTGGTCCGAGCTCGGATCCACTAGCTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTACCTGG PerlPrimer http://perlprimer.sourceforge.net/ for downloading Free open source standalone Runs in Windows, Linux, MacOS Features: Calculation of possible primer-dimers Retrieval of genomic or cDNA sequences from Ensembl (including both sequences automatically for Q-PCR) Ability to BLAST search primers using the NCBI server Results can be saved or optionally exported in a tab-delimited format that is compatible with most spreadsheet applications. ORF and CpG island detection algorithms Ability to add cloning sequences to primers, automatically adjusted to be in-frame Q-PCR primer design without manual intron-exon boundary entry PerlPrimer Other Resources NCBI: http://www.ncbi.nlm.nih.gov BLAST Entrez Genome Browser @ UCSC http://genome.ucsc.edu/ Genome Browser Human Mouse Rat Chimp In-Silico PCR Blat search SNPs Dog Chicken Fugu C. elegans Drosophila S. cerevisiae Special Applications Modified Oligonucleotides & Special Primers Degenerate Primers Mixed oligos e.g. actgattc[gc]tgct[atc] Nucleotides can be in unequal ratios Increased degeneracy means concentration of the individual primers decreases Deoxyinosine (dI) dI at degenerate positions rather than use mixed oligos dI base-pairs with any other base, effectively giving a four-fold degeneracy at any position in the oligo where it is present Degeneracies obviously reduce the specificity Autosticky PCR “dSpacer” protected tetrahydrofuran phosphoramidite For inclusion of abasic sites in an oligo Abasic sites cause stalling of DNA polymerases Can therefore be used to create 5’-overhangs in PCR products; “autosticky-PCR” Overhangs capable of annealing with restriction enzyme generated 5’-overhangs Chemical 5’-phosphorylation recommended Real Time PCR Considerations for primer design Smaller amplicon = higher efficiency amplicon ideally < 150 bp; maximum 400 bp Amplifying gDNA or cDNA gDNA: primers that are intron-specific cDNA: primers spanning exon-exon boundaries of spliced transcript Avoid a 3'-end T as this has a greater tolerance of mismatch Primer length: 18–30 nucleotides Taqman Probes Select the probe first and design the primers as close as possible to the probe without overlapping it Tm should be 68°–70°C No G on the 5´ end Select the strand that gives the probe more C than G bases Avoid runs of an identical nucleotide. This is especially true for guanine, where runs of four or more Gs should be avoided Fluorophore to quencher: optimally 6-14 bases apart Internally positioned quencher increases probe sensitivity Summary Oligo synthesis services that design Q-PCR primers and probes and guarantee them Many useful commercial programmes Multiple free tools for designing primers PerlPrimer (Desktop computer) : simple Primer3 (Web) : highly customisable CODEHOP (Web) : for degenerate primers Always check your primer sequence! Many published primers contain serious errors! The End That link again: http://users.bigpond.net.au/albert/primers.htm