primer design dalia 1

5/28/2018 Primer Design Dalia 1

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Primer Design

ByDr/ Dalia Shaalan

Lecturer of Medical Biochemistry

and Molecular BiologyMansoura University


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Why Are Primers Important?

Primers are what gives PCR itsSPECIFICITY!!!

Good primer design: PCR work is great.

Bad primer design: PCR work is terrible.


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Very-Brief PCR Reminder

PCR is a method to

amplify largequantities of a DNAcovering a specificsequence.


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PCR


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General notes on primer design in PCR

Perhaps the most critical parameter for

successful PCR is the design of primers

Primer selection

Critical variables that affect priming

are:- primer length

- specificity

- melting temperature (Tm)- G/C content

- 3-end sequence

- complementary primer sequences


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Primer length

Specificity and the temperature of annealing are dependenton primer lengthOligonucleotides between 20 and 30 bases are highlysequence specific

Primer length is inversly proportional to annealing efficiency:in general, the longer the primer, the more inefficient theannealingThe primers should not be too short as specificity decreases

Primer Size:

Too small, may bind to more than one site in the genome

Too Large (Long primers), takes a longer time to hybridize

and would slow down the PCR cycle.


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Factors affecting Tm:PRIMER LENGTH Longer primers stick better = melt at a

higher temperature.

GC CONTENT More G-C content = more triple bonds =

primers stick better = melt at highertemperature.

http://www.alkami.com/primers/refprmr.htm

Melting temperature (Tm)Not all primers will work the same; primers perform differently at

different temperatures. Melting temperature(Tm) is temperature at which the primer will

dissociate.


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Annealing temperature (Ta):

Optimal temperature for primers to attachto the template DNA

If you use too high Ta:

Bonds dont work

Primer doesnt anneal If you use too low Ta:

Primer may attach anywhere

Non-specific amplification

Depends on strength of bonds

Remember:

G-Cthree hydrogen bonds

A-Ttwo hydrogen bonds

Annealing temperature depends on GC content

Melting temperature (Tm)


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Melting temperature (Tm)the goal should be to design a primer with anannealing temperature of at least 50C.the relationship between annealingtemperature and melting temperature is one

of the Black Boxes of PCR.a general rule is to use an annealingtemperature that is 5C lower than themelting temperature.

PCR Annealing Temp = Melt T - 5C


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Melting temperature (Tm)both of the primers should be designed suchthat they have nearly similar meltingtemperatures.If primers are mismatched in terms of Tm,

amplification will be less efficient or may noteven work:

One primer with the higher Tm will mis-

prime at lower temperatures; Other primer with the lower Tm may not

work at higher temperatures.


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-A good working approximation of this value can be calculatedusing the Wallaceformula:

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

-The melting temperatures of oligos are most accuratelycalculated using nearest neighbor thermodynamiccalculations with the formula:

Tm= H [S+ R (c/4)] 273.15 C + 16.6 log 10[K+]

(H is the enthalpy, S is the entropy for helix formation, R isthe molar gas constant and c is the concentration of primer)

Melting temperature (Tm)


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G/C content

Ideally a primer should have a randommix of nucleotides and about 50% GCcontent.

There should be no PolyG or PolyCstretches that can promote non-specific

annealing.


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3-end sequence

The 3' terminal position in PCR primers isessential for the control of mis-priming.

Inclusion of a G or C residue at the 3'end of primers helps to ensure correctbinding (stronger hydrogen bonding = 3bonds of G/C residues).


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Complementary primer sequences

Primers need to be designed with absolutelyno intra-primer homologybeyond 3 basepairs. If a primer has such a region of self-homology, snap back can occur.

Another related danger is inter-primerhomology:partial homology in the middle

regions of two primers can interfere withhybridization. If the homology should occurat the 3' end of either primer, primer dimerformation will occur


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Complementarity

PRIMER-PRIMER (BAD) Excessive similarity between

primers, especially at the 3 ends,

leads to the formation of :

primer dimers

PRIMER-TARGET (GOOD)

Ideally should be 100% for

maximal specificity.

atcggactatcga

gctatacttatggcca

atcggactatcga

tagcctgatagctatacttatggcca


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What is a Primer-Dimer

An unwantedextension product

Results from primersannealing tothemselves, or eachother, at 3 ends

Extended primers areno longer available toprime target for PCR

atcggactatcga

gctatacttatggcca

atcggactatcgatatgaataccgga

tagcctgatagctatacttatggcca


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Two Strategies for Primer Design

?

Optimizing Primersfor Set Conditions:

Optimize the primerdesign to work in a

specific set of PCRconditions.

If youve got flexibilityaround the amplifiedsite.

Allows morestandardized PCRconditions.

Fixed Primers, VaryConditions:

Pick a primer pair andoptimize PCRconditions for it.

If an exact sequencesite needs to beamplified.

If youre workingwith someone elsesprimers.


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Strategy 1 for Primer Design:Fixed Primers, Vary ConditionsWith a given primer pair, the Tm can be calculated.

Run multiple PCR reactions, each using a differentannealing temperature (= Tm - 5).

Ta: 3C, -2C, -1C, 0C, +1C, +2C, +3C

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.


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Strategy 2 for Primer Design:Optimizing Primers for Set Conditions

PCR conditions (esp. annealingtemp) are kept constant.

Select primers for a theoreticalTm.

Best to select multiple primers,then experiment to see which

combination works best.

95C 65C72C

F1: atcgatcgatcgatcagtcatcg

F2: gtactgagctagctgcagctc

R1: atgactgagctgctagcttg

R2: atgcatgctcgtgactgtg

F1 F2

R1 F1/R1 F2/R1

R2 F1/R2 F2/R2


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Where do we get primer sequences from?

Somebody may have isolated them

Check databases

Freely available on internet (GenBank)

Results not publishable without primer

information Heterologous primers (from related species)

Primer design from published sequences.

Primer isolationVery lengthy and expensive procedure

several months work


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Primer design

Sequence should be reverse compliments

(i.e. 3 ends point toward one another).


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CATACGATGCTAGCTAGCTGCTAGTGCTG

ATCGTAGTCGTAGCTAGTCGTACGTACGT

CGATGTAATTCGCATCGATATGCGCTAGC

GATATGCGCATCGATCGATATCGATATGC

5

3

Primer 1

5-CATACGATGCTAGCT-3

Primer 2

3-GCTATAGCTATACG-5

Primer design

Primer 1: 5-CATACGATGCTAGCT-3 Primer 2: 5-GCATATCGATATCG-3


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CATACGATGCTAGCTAGCTGCTAGTGCTG

ATCGTAGTCGTAGCTAGTCGTACGTACGT

CGATGTAATTCGCATCGATATGCGCTAGC

GATATGCGCATCGATCGATATCGATATGC

5

3

Primer 1: 5-CTGCTAGTGC-3 Primer 2: 5-ATCGATGCGCAT-3

Primer design

Primer 1

5-CTGCTAGTGC-3

Primer 2

3-TACGCGTAGCTA-5


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PCR


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Designing Primers

Primer Design on the Web

Example: Primer3

Example gene: GFP5 Green Fluorescent Protein

GFP5, Genebank 1848286301 aggagaggac catcttcttc aaggacgacg ggaactacaa gacacgtgct gaagtcaagt 361 ttgagggaga caccctcgtc

aacaggatcg agcttaaggg aatcgatttc 1 ggatccaagg agatataaca atgagtaaag gagaagaact tttcactgga

gttgtcccaa 61 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg 121 aaggtgatgc

aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac 181 ctgttccatg gccaacactt gtcactactt

tctcttatgg tgttcaatgc ttttcaagat 241 acccagatca tatgaagcgg cacgacttct tcaagagcgc catgcctgag

ggatacgtgc aaggaggacg 421 gaaacatcct cggccacaag ttggaataca actacaactc ccacaacgta tacatcatgg 481

ccgacaagca aaagaacggc atcaaagcca acttcaagac ccgccacaac atcgaagacg 541 gcggcgtgca actcgctgat

cattatcaac aaaatactcc aattggcgat ggccctgtcc 601 ttttaccaga caaccattac ctgtccacac aatctgccct

ttcgaaagat cccaacgaaa 661 agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg 721

atgaactata caaataagag ctc

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


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Designing Primers

Primer Design on the Web Using Primer3

Enter sequence

Pick Primers


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Primer3 Output

Details:-Start-Length

-Tm-GC-Sequence

Designing Primers

Where they bind:


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Designing Primers

Primer 3 Output (continued)


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Primer Evaluation

Lets assume we selected the firstprimer pair (for + rev)

Website for online primer

evaluation:

TCATTGTTTGCCTCCCTGC

TAGAAACCCCAACCCGTGAAA

Enter Sequence


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Primer Evaluation

Website displays potential problemswith primer self-annealing

More advanced software can

examine interactions betweenprimers

TCATTGTTTGCCTCCCTGC

TAGAAACCCCAACCCGTGAAA

Graphical

Output


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Primer Evaluation

Just for fun, lets assume weselected a really BAD primer...

GGGCCCCTCACCAACCCGTGCCCGGG


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Live Example Primer DesignPrimer 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 brca1c. 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 sequencec. select options and choose Pick Primers

5. Analyse and double-check the primersa.http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.a

spxb. enter sequence, view

6. Order oligos.a. http://www.operon.com


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Conclusion on Primer design

Primer pairs should have similar annealing temp

length, %GC content

Tm = 4(G + C) + 2(A + T) oC.

Minimal (


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primer design dalia 1

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