Primer Design and Sequencing
ChE130 2015
General Rules
• Sterile technique!!
• Report any problems to TAs ASAP (and in advance if possible)
– Supply concerns (plates, media, tips, tubes, etc.)
– Bad reagents
• Take good notes
– This is critical for troubleshooting; help us help you.
• Cleanliness
– Keep enzymes (2X Pfu, 2X Paq, Restriction Enzymes, Ligase,
Phosphatase) on ice and put them away immediately after using
them.
– Double-check your work area before leaving
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Tips, etc in Biotrash
Dump liquid waste in fume hood
Gels in trash, pour running buffer into the bottle, rinse / dry running apparatus
Return materials to their proper locations
Turn off all machines (NanoVue, Gel Runners)
Don’t leave lids open
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Primer Design
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Primers
are short synthetic ssDNA or
oligonucleotides that serve as
starting points for DNA synthesis
“Forward” Primer:
5’ – GCCAGGAGTGAAACGATG – 3’
pYPET Template DNA:
5’ – GCCAGGAGTGAAACGATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGT — 3’
3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’
Denaturation
Annealing
5’ – GCCAGGAGTGAAACGATG — 3’
3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’
Elongation
5’ – GCCAGGAGTGAAACGATGTCTAAAGGTGAAGAA — 3’
3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’
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Primers
are short synthetic ssDNA or
oligonucleotides that serve as
starting points for DNA synthesis
“Reverse” Primer:
5’ – GCCACCTTGGCCTTAGTG – 3’
3’ – GTGATTCCGGTTCCACCG – 5’
pYPET Template DNA:
5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’
3’ – ACTTACTTAACATGTTTGTGGTGGTGGTGGTGGTGATTCCGGTTCCACCG — 5’
Denaturation
Annealing
5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’
3’ – GTGATTCCGGTTCCACCG — 5’
Elongation
5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’
3’ – GTGGTGGTGGTGGTGGTGATTCCGGTTCCACCG — 5’
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Guidelines for Primer Design
• Length
• Melting Temperature
– %GC Content
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Primer pair (length and Tm)
3’ GC Clamp
Extra nucleotides for restriction enzymes
No secondary structures
Avoid cross-homology
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Guidelines: Length
• Binding region (18-25 base pairs)
– A tradeoff between specificity and annealing
• Oligonucleotide synthesis are 99% efficient
– Max primer length will be 60bp
Length
% of Primers w/Correct Sequence
10 bases
20 bases
30 bases
40 bases
50 bases
(0.99)10 = 90.4%
(0.99)20 = 81.8%
(0.99)30 = 74.0%
(0.99)40 = 66.9%
(0.99)50 = 60.5%
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Guidelines: Melting Temperature (Tm)
• Tm is the temperature at which 50% of the
primer-target duplex dissociates
• Related to annealing temperature (Ta) for PCRs
– Aim for Ta = 55-60°C, which corresponds to Tm = 60-65°C
• Many online calculators
Can share PCR blocks if using
the same PCR program!
– Built-in to plasmid editors
– Online: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
• GC content 40%-60%
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Guidelines: Primer Pair
• Forward and reverse primers should have similar
length and Tm to:
– Avoid preferential amplification
– Simplify PCR optimization
• Aim for:
– Differences in length < 3 bases
– Differences in temperature < 3°C
Example, pYPet primers:
5’ – GCCAGGAGTGAAACGATG – 3’
5’ – GCCACCTTGGCCTTAGTG – 3’
Forward Primer: 18nt, Tm = 53.5°C
Reverse Primer: 18nt, Tm = 56.2°C
|Δlength| = 0
|Δtemperature| = 2.7°C
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Guidelines: Other Considerations
• 3’ GC Clamp
– The presence of a G or C at the 3’ end promotes correct
binding due to the stronger hydrogen bonding of G and C
bases
• Avoid runs and repeats
Restriction
Endonuclease
– Misprime
– Errors in synthesis
• Extra nucleotides for
DNA
restriction enzyme
(endonucleases) digestions
PDB: 1RVA
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pZE_Plac-YPet
Sequencing primer will be
provided for everyone
General Workflow
1. Use pZE_Plac-Ypet as both your
template DNA and as your
plasmid backbone
7 BamHI (1)
lac promoter 38..78
100 EcoRI (1)
RBS 110..122
121 KpnI (1)
seq_fwd 2886..2905
2. Design primers to amplify
Promoter and RBS region
YPet 127..861
AmpR 2800..1940
pZE_Plac-YPET
3005 bp
862 HindIII (1)
T1 893..936
seq_rev 1107..1088
T0 1827..1853
3. Use restriction enzyme (RE)
sites (BamH1, EcoR1, Kpn1, or
HindIII)
Note: these sites are unique,
double digest compatible and
give ‘sticky ends’
4. Create designed plasmid
through ligation at RE sites
colE1 996..1803
lacI binding site
colFWD
RBS
lac promoter
BamHI -35 box
Seq FWD
-10 box
colREV1
EcoRI
KpnI
colREV2
YPET
HindIII
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Possible Cloning Strategies
RBS Modifications
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RBS
lac promoter
BamHI
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-35 box
-10 box
EcoRI
BamHI
-35 box
-10 box
EcoRI
HindIII
KpnI
RBS
lac promoter
YPET
KpnI
YPET
HindIII
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Possible Cloning Strategies
Promoter Modifications
Changes in
the -35 box
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RBS
lac promoter
BamHI
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-35 box
-10 box
EcoRI
BamHI
-35 box
-10 box
EcoRI
HindIII
KpnI
RBS
lac promoter
YPET
YPET
HindIII
KpnI
BamHI
EcoRI
Changes in sequence
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Possible Cloning Strategies
Other Methods
QuickChange
NNNNNN
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RBS
lac promoter
BamHI
-35 box
-10 box
EcoRI
YPET
HindIII
KpnI
NNNNNN
Error-Prone PCR
BamHI
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RBS
lac promoter
BamHI
-35 box
-10 box
EcoRI
KpnI
YPET
HindIII
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Sequencing
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How does sequencing work?
Plasmid DNA
sample
Sequencing primer is located ~100bp
upstream of segment to be sequenced
• Sequencing results are not accurate
within the first 100bp region
• Accuracy also tapers off after 800900bp
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Sequencing Plasmid DNA
• Miniprep overnight culture of cells harboring desired plasmid
• Measure concentration of plasmid DNA
– Check A260/280
– Verify samples are plasmid DNA
• Carefully label tubes for samples to be sent for sequencing
– At least 400 ng of plasmid
– 10 µL of 10 μM sequencing primer
• If submitting more than 3 sample, add 2μL sequencing primer for each
additional sample
• Prepare online form
– Enclose tubes and printout in a plastic bag
• Dropbox will be located outside of Braun 16/17
– Pickup is at 1pm for Retrogen
– Pickup is at 9am and 3pm for Laragen
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But before you sequence…
• Verify plasmid DNA is plasmid DNA
and contains insert of correct length
– Colony PCR
– Restriction mapping
– DNA gel electrophoresis
+Insert
seq_fwd 2886..2905
7 BamHI (1)
lac promoter 38..78
100 EcoRI (1)
RBS 110..122
121 KpnI (1)
No Insert
YPet 127..861
AmpR 2800..1940
pZE_Plac-YPET
3005 bp
862 HindIII (1)
T1 893..936
seq_rev 1107..1088
T0 1827..1853
DNA gel
colE1 996..1803
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Go to http://sequencing.retrogen.com
Or: http://sequencing.laragen.com/
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Home Screen
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Submit sequencing requests - Retrogen
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Submit sequencing requests - Laragen
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Home Screen
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Results, next morning by 10am
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DNA editing software
Key features:
restriction site recognition, DNA annotations
• ApE (A plasmid Editor)
– OS X and Windows
http://biologylabs.utah.edu/jorgensen/wayned/ape/
• pDRAW32
– Windows only
http://www.acaclone.com/
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