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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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Recommended