Pcr primer design and marker development by Magdy Mohamed

PCR primer design and marker development

Contact: Marwa Mahmoud Ghonaim marwa_ghonaim@yahoo.com

Polymerase Chain Reaction PCR primer design is the creation of short nucleotide sequences for use in amplifying region of DNA (random or specific regions):  Amplifies a single or a few copies of DNA molecular to generate up to billion of copies of a particular DNA sequence.  PCR is most common and often indispensable technique used in molecular biological research labs for a variety of applications.

PCR Method

The polymerase chain reaction (PCR) is a method by which DNA is amplified Method for exponential amplification of DNA sequences Basic requirements: – Template: DNA or RNA; – One or two DNA-oligonucleotides (also called DNA primers), are complementary to either end of the target sequence but lie on opposite strands; – Thermostable DNA polymerase (Taq, Tth, Pfu polymerases);

– Desoxynucleotides (dATP, dTTP, dCTP, dGTP) and appropriate reaction buffer;

The DNA synthesis and exonuclease activities of DNA polymerases

Temperature cycling 3 step process: • Annealing temperature (usually 37-68°C) - primers hybridize to template • Extension temperature (usually 68-72°C) - DNApolymerase synthesized new DNA chain from primer • Denaturation temperature (usually 94-98°C) separates complemented DNA strands

• Process repeated for approximately 20 to 40 cycles

Polymerase Chain Reaction

Why are primers important?

• Primers are what gives PCR its SPECIFICITY! • Good primer design: PCR works correctly • Bad primer design: PCR works not efficient

PCR primers are designed to:  Highly conserved DNA regions  Protein-coding regions with low degeneracy  More conserved regions that flank variable regions

PCR Primer Design

ď&#x192;źPrimer design is a critical step in all types of PCR methods to ensure specific and efficient amplification of target sequence so it is the key for successful PCR.

General primer design guidelines Primer Specificity primers must be specific for desired sequence (conserved nucleotide or protein regions) to be amplified; Primer length primers should be long enough to ensure specificity (usually 18-30 bases), If the length is too short, it is difficult to design gene-specific primers and choose optimal annealing temperature;

• Good primer must be:

• no complementarity between forward and reverse primers or primers and product. • Melting temperature (55-80°C). Base composition • G+C content should be between 40% and 80%, with an even distribution of all four bases along the length of the primer.

PCR primers design factors Characteristics of primers: Specificity Specific for the intended target sequence (avoid nonspecific hybridization)

Thoughts on primer design: Uniqueness Linguistic Complexity Length

Stability Forms table duplex with template under PCR conditions

Melting Temperature Annealing Temperature Stability at the 3â&#x20AC;˛ end in primer

Compatibility Primers used as a pairs shall work under the same PCR conditions

Primer Pair Matching

PCR primers design factors 1. Primer efficiency - of a reasonably high Tm, - without dimers, especially on their 3’-ends (to prevent self-extension), - without hairpin stems, especially on their 3’-ends (to prevent selfpriming), - Avoid repetitive sequences to ensure quick and correct annealing. - all primers in one incubation mixture should not form significant 3’dimers between each other. 2. High specificity - long enough to increase specificity, - unique, especially at its 3’-end, to avoid false priming, - moderately stable at its 3’-end (as opposed to highly GC-rich) to ensure that a very short fragment won’t initialize the extension (too low 3’-end stability hurts the priming efficiency).

Sequence linguistic complexity (LC) linguistic complexity : nucleotides arrangement and composition Linguistic complexity (LC) values for sequence length (s) are converted to percentages, in which 100% means maximal ‘vocabulary richness’ of a sequence: Primer sequence

LC, %

5’-AAAAAAAAAAAAAAAAAAAAA

5’-ACACACACACACACACACACA

5’-TTTTTTTTTTGGGGGGGGGAG

5’-GCTACCAATGAGAAGGTCACGT

5’-TGTTCTCCCATAGCACAAGAGGA

5’-TGGCTATTCTGAACCAGCGTTGC

100

Uniqueness

ď&#x201A;§optimal primers should hybridize only to the target sequence, particularly when complex genomic DNA is used as the template. ď&#x201A;§Amplification problems can arise due to primers annealing to repetitious sequences (retrotransposons, DNA transposons, or tandem repeats).

ď&#x192;&#x2DC;To improve Uniqueness avoid regions of homology. Primers designed for a sequence must not amplify other genes in the mixture. ď&#x192;&#x2DC;You can BLAST the templates against the appropriate database. It will identify regions with significant cross homologies in each template and avoid them during primer search.

Length Primer length has effects on uniqueness and

melting/annealing temperature: â&#x20AC;˘ the longer the primer, the more chance that itâ&#x20AC;&#x2122;s unique (18-30 bases) â&#x20AC;˘ the longer the primer, the higher melting/annealing temp.

3’ Stability & 5’ Stability: Primer elongation starts at 3’ end. Therefore, once the 3’ end hybridizes to the template stably, the elongation begins. 5’ end sequence plays less important role. The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. Ideal situation: More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

Melting temperature (Tm) calculation the temperature in degrees Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands. Factors affecting Tm: Concentration of DNA. Concentration of ions in the solution, most notably Mg+ and K+.

DNA sequence. Length of DNA.

Generally, sequences with higher fraction of GC base pairs, have a higher Tm than do AT-rich sequences.

The simplest equation based on base content is the “Wallace rule” (where L is the length of the hybrid duplex in base pairs):

Tm ( C )  2( L  G  C ) 

Melting temperature (Tm) calculation

The stability of the DNA double helix depends on a fine balance of interactions including hydrogen bonds between bases. Base-stacking interactions increase with increasing salt concentration, as high salt concentrations mask the destabilising charge repulsion between the two negatively charged phosphodiester backbones. DNA duplex stability therefore increases with increasing salt concentration. Divalent cations such as Mg2+ are more stabilising than Na+ ions, and some metal ions bind to specific loci on the DNA duplex.

Annealing Temperature (Ta) calculation The range of temperatures where efficiency of PCR amplification is maximal without non-specific products. Generally, you should use an annealing temperature about 5Â°C below the Tm of your primers. The optimal annealing temperature (Ta Opt) for any given primer pair on a particular target can be calculated as follows:

Ta = 0.3 x Tm(primer) + 0.7 Tm (product) â&#x20AC;&#x201C; 14.9 where, Tm(primer) = Melting Temperature of the primers Tm(product) = Melting temperature of the product

Too high Ta

primer-template hybridization

low PCR

product yield.

Too low Ta

non-specific products

caused by a high

number of base pair mismatches,. Mismatch tolerance have the strongest influence on PCR specificity.

Annealing Temperature (Ta) calculation Gradient of PCR annealing Tm: 45 - 58 C

Tm Taopt Taopt-Tm

40 52 12

41 54 13

49 >58 ~10

45 56 11

53 >58 ~10

55 >58 ~10

CG%

50%

67%

58%

75%

Secondary Structure ď śThe Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. Hairpins: It is formed by intramolecular interaction within the primer and should be avoided.

ď&#x192;&#x2DC;

Self Dimer: formed by intermolecular interactions between the two (same sense) primers, where the primer is homologous to itself.

ď&#x201A;§

Generally a large amount of primers are used in PCR compared to the amount of target gene. When primers form intermolecular dimers much more readily than hybridizing to target DNA, they reduce the product yield.

ď&#x192;&#x2DC; Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous.

Intra-molecular interactions will give rise to hairpins inter-molecular hybridization will give rise to dimers.

Primer dimer detection criteria (A–C) Interactions between primers; (D) Hairpin structures; (E) Undesirable binding of primers to template sequence.

Primer-dimers involving one or two sequences may occur in a PCR reaction. Stable primer dimer formation is very effective at inhibiting PCR since the dimers formed are amplified efficiently and compete with the intended target.

What is a primer-dimer

3â&#x20AC;&#x2122;-end dimer:

5â&#x20AC;&#x2122;-end and internal dimers:

Complementarity • PRIMER-PRIMER – Excessive similarity between primers, especially at the 3’ ends, leads to the formation of “primer dimers” • PRIMER-TARGET – Ideally should be 100% similar for maximal specificity. – Primers don’t HAVE to be perfectly similar to target to work.

ď&#x192;&#x2DC; Repeats:

A repeat is a di-nucleotide occurring many times

consecutively and should be avoided because they can misprime. For example: ATATATAT. A maximum number of di-nucleotide repeats

acceptable in an oligo is 4 di-nucleotides. ď&#x192;&#x2DC;Runs: Primers with long runs of a single base should generally be

avoided as they can misprime. For example, AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A maximum number of runs accepted is 4bp.

for Primer Design: Fixed Primers, Vary Conditions • With a given primer pair, the Tm can be calculated. • Run multiple PCR reactions, each using a different annealing temperature (= Tm - 5). • “Bracket” Ta: – 10C, -5C, 0C, +5C, +10C • Temp too low: Smearing due to non-specific priming • Temp too high: No amplification due to no priming • Choose conditions which give the best results.

Web sites related to PCR primer designer

PrimerDigital online tools: http://primerdigital.com/tools/ NCBI/Primer-BLAST (Primer3): http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Oligomer online PCR tools: http://www.oligomer.fi/en/analyysityokalut IDT online SciTools: http://idtdna.com/scitools/scitools.aspx

MWG /Operon tools: http://www.eurofinsgenomics.eu/

sequence

PrimerID

Sequence(5'-3')

Tm(째C)

Primer_Quality(%)

PCR_Fragment_Size(bp)

1F33_1_651-671

tacctctggggagcaacttgg

59.2

1R83_1_756-777

gtagctgatgaactcggagtgc

57.5

1F39_1_745-764

ctgatcaagaagcactccga

54.7

1R35_1_1788-1807

ccttgatgaccttgcagagg

55.8

1F39_1_745-764

ctgatcaagaagcactccga

54.7

1R27_1_1938-1957

acccagacatgctggagtcc

59.5

1F47_1_1013-1033

tctacaagagcttgaccaacg

54.9

1R73_1_1046-1065

gaagtgcttcacggcaagat

56.2

1F47_1_1013-1033

tctacaagagcttgaccaacg

54.9

1R35_1_1788-1807

ccttgatgaccttgcagagg

55.8

1F47_1_1013-1033

tctacaagagcttgaccaacg

54.9

1R27_1_1938-1957

acccagacatgctggagtcc

59.5

1F48_1_1039-1058

gaggagcatcttgccgtgaa

57.9

1R54_1_1419-1438

tggagtcctcgtggatgcca

61.2

1F48_1_1039-1058

gaggagcatcttgccgtgaa

57.9

1R35_1_1788-1807

ccttgatgaccttgcagagg

55.8

1F48_1_1039-1058

gaggagcatcttgccgtgaa

57.9

1R30_1_1893-1912

gctccatgttggcggtccaa

61.1

1F48_1_1039-1058

gaggagcatcttgccgtgaa

57.9

1R27_1_1938-1957

acccagacatgctggagtcc

59.5

Topt(째C)

127

1063

1213

795

945

400

769

874

919

Primer Evaluation

Primer list analysis

nt A T C G GC% Tm°C Molecular Weight(g/mole) nmol µg/OD260 Linguistic_Complexity(%) Primer's_PCR_Efficiency(%)

Name

Sequence

1f33_1_651-671

tacctctggggagcaacttgg

21 4 5 5 7

57.1

59.2

6462.2

5.1

32.7

1r83_1_756-777

gtagctgatgaactcggagtgc

22 5 5 4 8

54.5

57.5

6815.5

4.6

31.6

1f39_1_745-764

ctgatcaagaagcactccga

20 7 3 6 4

54.7

6095

5.1

30.9

1r35_1_1788-1807 ccttgatgaccttgcagagg

20 4 5 5 6

55.8

6133

5.3

32.6

1f39_1_745-764

ctgatcaagaagcactccga

20 7 3 6 4

54.7

6095

5.1

30.9

1r27_1_1938-1957 acccagacatgctggagtcc

20 5 3 7 5

59.5

6087

5.2

31.9

1f47_1_1013-1033 tctacaagagcttgaccaacg

21 7 4 6 4

47.6

54.9

6399.2

4.9

31.3

1r73_1_1046-1065 gaagtgcttcacggcaagat

20 6 4 4 6

56.2

6166.1

5.1

31.1

1f47_1_1013-1033 tctacaagagcttgaccaacg

21 7 4 6 4

47.6

54.9

6399.2

4.9

31.3

1r35_1_1788-1807 ccttgatgaccttgcagagg

20 4 5 5 6

55.8

6133

5.3

32.6

1f47_1_1013-1033 tctacaagagcttgaccaacg

21 7 4 6 4

47.6

54.9

6399.2

4.9

31.3

1r27_1_1938-1957 acccagacatgctggagtcc

20 5 3 7 5

59.5

6087

5.2

31.9

1f48_1_1039-1058 gaggagcatcttgccgtgaa

20 5 4 4 7

57.9

6182.1

5.1

31.3

1r54_1_1419-1438 tggagtcctcgtggatgcca

20 3 5 5 7

61.2

6149

5.3

32.6

1f48_1_1039-1058 gaggagcatcttgccgtgaa

20 5 4 4 7

57.9

6182.1

5.1

31.3

1r35_1_1788-1807 ccttgatgaccttgcagagg

20 4 5 5 6

55.8

6133

5.3

32.6

1f48_1_1039-1058 gaggagcatcttgccgtgaa

20 5 4 4 7

57.9

6182.1

5.1

31.3

1r30_1_1893-1912 gctccatgttggcggtccaa

20 3 5 6 6

61.1

6109

5.4

33.1

1f48_1_1039-1058 gaggagcatcttgccgtgaa

20 5 4 4 7

57.9

6182.1

5.1

31.3

1r27_1_1938-1957 acccagacatgctggagtcc

20 5 3 7 5

59.5

6087

5.2

31.9

Primer Evaluation • Let’s assume we selected the first primer pair (for + rev) • Website for online primer evaluation:

Enter Sequence

TCATTGTTTGCCTCCCTGC TAGAAACCCCAACCCGTGAAA

Live Example Primer Design Primer Design Workflow: 1. Pick a gene. ie. BRCA1 2. Pull up sequence for the gene. a. http://www.ncbi.nlm.nih.gov/ b. search Nucleotide Database for brca1 c. scroll through accessions for desired one 3. Copy sequence to text editor. 4. Pull up a primer design website. a. http://frodo.wi.mit.edu/ b. copy sequence c. select options and choose Pick Primers 4b. Verify primers find target (optional) a. http://www.ncbi.nlm.nih.gov/BLAST/ b. select nucleotide blast c. enter primer sequence, choose blast 4c. Analyse and double-check the primers a. http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx b. enter sequence, view 5. Order oligos. a. http://www.operon.com

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

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

3.4 PCR Primer Design

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

The Primer

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

Generator 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 Primâ&#x20AC;&#x2122;er

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

Project

ect/primer.html

GAP

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

Software name Primerselect

Description Analyses a template DNA sequence and chooses primer pairs for PCR and primers for DNA sequencing

DANSIS Max

DANASIS Max is a fully integrated program that includes a wide range of standard sequence analysis features.

Primer Primer 5

Primer design for windows and power macintosh.

Primer Primer:

Comprehensive primer design for windows and Power Macintosh.

NetPrimer

Comprehensive analysis of individual primers and primer pairs.

Array Designer 2

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

AlleleID 7

Design molecular beacons and TaqMan probes for robust amplification and fluorescence in real time PCR.

GenomePRIDE 1.0

Primer design for DNA-arrays/chips.

Fast PCR

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.

OLIGO 7

Primer Analysis Software for Mac and Windows.

Primer Designer 4

Will find optimal primers in target regions of DNA or protein molecules, amplify leatures in molecules, or create products of a specified length.

GPRIME

Software for primer design.

Sarani Gold

Genome Oligo Designer is a Software for automatic large scale design of optimal oligonucleotide probes for microarray experiments.

PCR Help

Primer and template design and analysis.

Genorama chip Design

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

Software

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

Primer Designer

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 Primer

Automatic design tools for PCR. Sequencing or hybridization probes, degenerate primer design, restriction, Nested/Multiplex primer design, restriction enzyme analysis and more.

PreimerDesign

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

How we use primers in Retro-markers

There are several techniques using retroelements as molecular markers:

 S-SAP (Sequence-Specific Amplified Polymorphism)  IRAP (Inter Retrotransposon Amplified Polymorphism)  REMAP (REtrotransposon Microsatellite Amplification polymorphisms)  RBIP (Retrotransposons-Based Insertion Polymorphism)  iPBS (inter Primer Binding Sites amplification)

iPBS (inter Primer Binding Sites amplification)