in the name of god. pcr primer design lecturer: dr. farkhondeh poursina

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IN THE NAME OF GOD

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IN THE NAME OF GOD

PCR Primer DesignPCR Primer Design

Lecturer: Dr. Farkhondeh PoursinaLecturer: Dr. Farkhondeh Poursina

DefinitionDefinition

PCR primer design is the creation of short nucleotide sequences for use in amplifying a specific region of DNA.

Polymerase chain reaction (PCR)

Developed in 1985 by Kary Mullis

Amplifies a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA Sequence.

PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.

ExamplesExamples

PCR primers are designed to:

Highly conserved DNA regionsProtein-coding regions with low

degeneracyMore conserved regions that flank variable

regions

ApplicationsApplications

Primer design is used for:

Finding new genesDeveloping new identification tools

Optimizing PCRsCloning Sequencing, DNA-based phylogeny, functional analysis of genes etc….

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Primer Design CriteriaPrimer Design Criteria

Primer Specificity

Target: Conserved nucleotide or protein regions

Primer lengthIf the length is too short, it is difficult to design gene-specific

primers and choose optimal annealing temperature. On the other hand, longer primers are more likely to form secondary structures that result in decreased PCR efficiency or promote primer dimer formation ,,Usually 18 - 24 bases

Base compositionG+C content should be between 40% and 60%, with an even

distribution of all four bases along the length of the primer.9

End with 1-2 GC pairs, if possible,Primer 3’ end stability

No inter- or intra-primer interactions Check with databases for specificityCycling conditions and buffer concentrations should be

adjusted for each primer pair (see PCR troubleshooting)

Avoiding base run.

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Avoid sequence secondary structures

Avoid complementary at 3` end of primers

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Primer melting temperature (Tm):

Tm: is the temperature at which half the DNA strands are single stranded and half are double-stranded.

The melting temperature (Tm) is the most important factor in determining the optimal PCR annealing temperature (Ta).

Calculated Tm values of members of a primer pair should not differ by >5°C.

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Wallace rule:

Tm = 4 * (G + C) + 2 * (A + T)

Bolton and McCarthy:

Tm = 81.5 + 16.6 * Log [I] + 0.41 * (%GC) – 600/L

The nearest neighbor method (Santalucia et.al, 1998):

Δ G (Gibbs free energy) is the most important factor that is to be taken into consideration in primer designing.

Δ G definition: The Gibbs Free Energy G is the measure of the amount of work that can be extracted from a process operating at a constant pressure. It is the measure of the spontaneity of the reaction.

Primer secondary structures: Presence of the primer secondary structures adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.

Hairpins: A 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/mol is tolerated generally.

Larger negative value for ΔG indicates stable, undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.

Self Dimer : A 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally.

Rules for Primer designing…….

Cross Dimer: Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and

an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.

3' End Stability : It is the maximum ΔG value (-8.5 kCal/mol is the default,

however a value of -10 to -12kCal/mol is tolerated) of the five bases from

the 3’.

An unstable 3' end (less negative ΔG) will result in less false priming.

Primers with pentamer ΔG more stable than -12.0 kCal/mol (more

negative) have a tendency to false prime and are more likely to form

hairpins and self dimers.

Rules for Primer designing…….

Primer Design SoftwaresPrimer Design Softwares

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Tool name URL

CODEHOP http://blocks.fhcrc.org/codehop.html

Gene Fisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/

DoPrimer http://doprimer.interactiva.de/

Primer3 http://frodo.wi.mit.edu/primer3/

Primer Selection Http://alces.med.umn.edu/rawprimer.html

Web Primer http://genome.www2.stanford.edu/cgi.bin/SGD/web.primer

PCR designer http://cedar.genetics.ston.ac.uk/public_html/primer.html

Primo pro 3.4 http://www.changbioscience.com/primo.html

Primo Degenerate

3.4

http://www.changbioscience.com/primo/primod.html

PCR Primer Design http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer

The Primer

Generator

http://www.med.jhu.edu/medcenter/primer/primer.cgi

EPRIMERS http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html

PRIMO http://bioweb.pasteur.fr/seqanal/interfaces/eprimo.html3

PrimerQuest http://www.idtdna.com/biotools/primer_quest/primer_quest.asp

MethPrimer http://itsa.uscf/~uralab/methprimer/index1.html

Rawprimer http://alces.med.umn.edu/rawprimer.html

MEDUSA http://www.cgr.ki.se/cgr/MEDUSA/

The Primer Prim’er

Project

http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj

ect/primer.html

GAP http://promoter.ics.uci.edu/primers/

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Description Software name

Analyses a template DNA sequence and chooses primer pairs for PCR and

primers for DNA sequencing

Primerselect

DANASIS Max is a fully integrated program that includes a wide range of

standard sequence analysis features.

DANSIS Max

Primer design for windows and power macintosh. Primer Primer 5

Comprehensive primer design for windows and Power Macintosh. Primer Primer:

Comprehensive analysis of individual primers and primer pairs. NetPrimer

For fast, effective design of specific oligos or PCR primer pairs for microarrays. Array Designer 2

Design molecular beacons and TaqMan probes for robust amplification and

fluorescence in real time PCR.

AlleleID 7

Primer design for DNA-arrays/chips. GenomePRIDE 1.0

Software for Microsoft Windows has specific. Ready-to-use template for many

PCR and sequencing applications; standard and long PCR inverse PCR.

Degenerate PCR directly on amino acid sequence. Multiplex PCR.

Fast PCR

Primer Analysis Software for Mac and Windows. OLIGO 7

Will find optimal primers in target regions of DNA or protein molecules, amplify

leatures in molecules, or create products of a specified length.

Primer Designer 4

Software for primer design. GPRIME

Genome Oligo Designer is a Software for automatic large scale design of optimal

oligonucleotide probes for microarray experiments.

Sarani Gold

Primer and template design and analysis. PCR Help

Genorama Chip Design Software is a complete set of programs required for

genotyping chip design.The programs can also be bought separately.

Genorama chip Design

Software

The Primer Designer features a powerful, yet extremely simple, real-time interface

to allow the rapid identification of theoretical ideal primers for your PCR

reactions.

Primer Designer

Automatic design tools for PCR. Sequencing or hybridization probes, degenerate

primer design, restriction, Nested/Multiplex primer design, restriction enzyme

analysis and more.

Primer Primer

DOS-program to choose primer for PCR or oligonucleotide probes. PreimerDesign

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Gene of interest

Mutation Detection

Allelic discrimination

Gene expression ( mRNA)

Microbial agents detection

Quantification

… Disease

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