primer design final

7/29/2019 Primer Design FInal

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What is a primer?hat is a primer?A primer is a short synthetic oligonucleotide which

is used in many molecular techniques from PCR to

Important considerations on primer design1. Primers should be 17-30 bases in length

2. Base com osition should be 40-60 % G+C

3. Primers should end (3') in a G or C, or CG or GC:

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.

5. Tm difference between forward and reverse

pr mers s ou e

6. 3'-ends of primers should not be

complementary (ie. base pair), as otherwise

primer dimers will be synthesized preferentially

to any other product

7. Primer self-com lementarit abilit to form

2o structures such as hairpins) should be

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Practical consideration for primer pair design1. Amplicon Length: The amplicon length is dictated bythe experimental goals. If you know the positions of

each primer with respect to the template, the product is

calculated as:

Product length = (Position of antisense primer - Position

of sense primer) + 1.2. Product Position: Primer can be located near the 5'end, the 3' end or any where within specified length.

enera y, e sequence c ose o e en s nown

with greater confidence and hence preferred most

.

Cont3


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3. Tm of Product: Melting Temperature (Tm) is thetemperature at which one half of the DNA duplex

will dissociate and become single stranded. The

stability of the primer-template DNA duplex can be

measured by the melting temperature (Tm).

4. Tm Calculation:Tm = 4(G+C) + 2(A+T) CWhere, G+C is the total GC content in the primer,

A+T is the total AT content in the primer

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Note: 1. The actual T is influenced b theconcentration of Mg2+, K+, and co-solvents

2. The formula given above for (Tm) is

simplistic; there are many primer design

programs which use more complex nearest-

neighbor thermodynamics values

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5. Annealing temperature: The annealing temperaturea c osen or a epen s rec y on eng an

composition of the primer(s). Generally, an annealing

pair of primers is used

6. Optimal annealing temperature calculation:. Optimal annealing temperature calculation:optimal annealing temperature for any given primer

air on a articular tar et can be calculated as

follows:

Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) 25Where, Tm of primeris the melting temperature of the

less stable primer-template pair and Tm of product is

the melting temperature of the PCR product.

Cont6


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Primer designing for cloning purposeGeneral considerations:1. All commonly-used cloning/expression vectors

contain a multiple cloning site (MCS) with different

restriction sites

2. When designing your primers, you should add one of

ese res r c on s es o e en o your pr mer n suc

as was as to preserve the reading frame of the target

.

3. Choose a restriction site that is not present in the gene

4. Add 3-4 extra G, C or GCs at the end of the restriction

sites, as it is necessary (depending the restriction sites)

for restriction enzyme to cleave the restriction site.

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Example

Primer design for the PCR amplification of the cut1 geneThe

cut1gene from

Thermobifida fuscaencodes for cutinase, an

enzyme involved in the hydrolysis of plant cutin.

The cut1 gene:>cut1

ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCAAT

GGTCATATCGATATCGTGACGCGCGCCACGCAGATGTTCGATCACGTT

ATTCTGGCGATTGCCGCCCCGCAGCGACGATTGCAGTCACAGCAGGC

AACCGCGCATCTGGGGAACGTGGAAGTGGTCGGGTTTAGTGATTTAA

TGGCGAACTTCGCCCGTAATCAACACGCTACGGTGCTGATTCGTGGC

AATCGCCACTTAATGCCGGAACTGGAAAGTGTGTTTCTGATGCCGTCG

AAAGAGTGGTCGTTTATCTCTTCATCGTTGGTGAAAGAGGTGGCGCG

CCATCAGGGCGATGTCACCCATTTCCTGCCGGAGAATGTCCATCAGGC

8GCTGATGGCGAAGTTAGCG

Cont


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Suppose, we decided to clone the gene into an expression vector using the

restriction enzymes EcoRI (5'-end) and BamHI (3'-end).Here we show the design of both primers:

5' d i'-end primerThe EcoRI site in the vector is in frame with the ATG in the gene to createthe N-terminal methionine residue of CUT1.

5-CATGGGATCCATGCAAAAACGGGCGATTTATCC-35' extension (CATG)

EcoRI restriction site (GGATCC)Start codon (ATG)ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCAATGGTCATATCGATATCGTGACGCGCGCCACGCAGATGTTCGATCACG

TTATTCTGGCGATTGCCGCCCCGCAGCGACGATTGCAGTCACAGCAG

9 ContTAATGGCGAACTTCGCCCGTA


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3'-end primerC-terminal His tag is being used, so we have to delete stop codon

(TAG, the natural stop codon for CUT1 to get a fusion of cutinase 1

rotein with C-terminal His ta .

5-CGGGATCCCGCTAACTTCGCCATCAGC-35' extension (CG)

BamH I restriction site (GGATCC)

ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACCA

.GCCATCAGGGCGATGTCACCCATT

TCCTGCCGGAGAATGTCCATCAGGCGCTGATGGCGAAGTTAGCG

5' GCTGATGGCGAAGTTAGCG 3'3' CGACTACCGCTTCAATCGC 5

5' CGCTTAACTTCGCCATGAGC 3'

10 Cont5 CGCTTAACTTCGCCATGAGC 3


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Primer binding pattern with gene

5-CATGGGATCCATGCAAAAACGGGCGATTTATCC-3

ATGCAAAAACGGGCGATTTATCCGGGTACTTTCGATCCCATTACC

AATGGTCATATCGATATCGTGACGCGCGCCACGCAGATGTTCGATCACGTTATTCTGGCGAT

TGCCGCCCCGCAGCGACGATTGCAGTCACAGCAGGCAACCGCGCATCTGGGGAACGTGG

AAGTGGTCGGGTTTAGTGATTTAATGGCGAACTTCGCCCGTAATCAACACGCTACGGTGCT

GATTCGTGGCCTGCGTGCGGTGGCAGATTTTGAATATGAAATGCAGCTGGCGCATATGAAT

CGCCACTTAATGCCGGAACTGGAAAGTGTGTTTCTGATGCCGTCGAAAGAGTGGTCGTTTA

TCTCTTCATCGTTGGTGAAAGAGGTGGCGCGCCATCAGGGCGATGTCACCCATTTCCTGCC

GGAGAATGTCCATCAGGCGCTGATGGCGAAGTTAGCG

3-CGACTACCGCTTCAATCGCCCTAGGGC-5

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Some useful primer designing softwares

Primer3

r mer-

Fast PCR

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primer design final

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