Primer Design and Sequencing

ChE130 2015

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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

• 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

Denaturation Annealing

5’ – GCCAGGAGTGAAACGATG – 3’

“Forward” Primer:

5’ – GCCAGGAGTGAAACGATG — 3’3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’

5’ – GCCAGGAGTGAAACGATGTCTAAAGGTGAAGAA — 3’3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’

Elongation

5’ – GCCAGGAGTGAAACGATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGT — 3’3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’

pYPET Template DNA:

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Primersare short synthetic ssDNA or oligonucleotides that serve as starting points for DNA synthesis

Denaturation Annealing

Elongation

pYPET Template DNA:

5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’ 3’ – GTGGTGGTGGTGGTGGTGATTCCGGTTCCACCG — 5’

5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’ 3’ – GTGATTCCGGTTCCACCG — 5’

5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’3’ – ACTTACTTAACATGTTTGTGGTGGTGGTGGTGGTGATTCCGGTTCCACCG — 5’

5’ – GCCACCTTGGCCTTAGTG – 3’

“Reverse” Primer:

3’ – GTGATTCCGGTTCCACCG – 5’

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Guidelines for Primer Design

• Length• Melting Temperature– %GC Content

• 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

Length10 bases20 bases30 bases40 bases50 bases

% of Primers w/Correct Sequence(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– Built-in to plasmid editors– Online: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

• GC content 40%-60%

Can share PCR blocks if using the same PCR program!

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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

5’ – GCCAGGAGTGAAACGATG – 3’Forward Primer: 18nt, Tm = 53.5°C

5’ – GCCACCTTGGCCTTAGTG – 3’Reverse Primer: 18nt, Tm = 56.2°C

|Δlength| = 0|Δtemperature| = 2.7°C

Example, pYPet primers:

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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– Misprime– Errors in synthesis

• Extra nucleotides for restriction enzyme (endonucleases) digestions

PDB: 1RVA

Restriction Endonuclease

DNA

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pZE_Plac-YPet General Workflow1. Use pZE_Plac-Ypet as both your

template DNA and as your plasmid backbone

2. Design primers to amplify Promoter and RBS region

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

Sequencing primer will be provided for everyone

YPETRBSlac promoter

lacI binding site colREV1 colREV2colFWD

Seq FWD

-35 box -10 boxBamHI EcoRI KpnI HindIII

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Possible Cloning StrategiesRBS Modifications

BamHI

KpnI

RBS

YPETRBSlac promoter-35 box -10 boxBamHI EcoRI KpnI HindIII

YPETRBSlac promoter-35 box -10 boxBamHI EcoRI KpnI HindIII

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RBS

EcoRI

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Possible Cloning StrategiesPromoter Modifications

YPETRBSlac promoter-35 box -10 boxBamHI EcoRI KpnI HindIII

YPETRBSlac promoter-35 box -10 boxBamHI EcoRI KpnI HindIII

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BamHI

Changes in the -35 box

BamHIEcoRI

Changes in sequence

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Possible Cloning Strategies

YPETRBSlac promoter-35 box -10 boxBamHI EcoRI KpnI HindIII

YPETRBSlac promoter-35 box -10 boxBamHI EcoRI KpnI HindIII

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6BamHI

NNNNNN

NNNNNN

QuickChange

Error-Prone PCR

Other Methods

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Sequencing

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How does sequencing work?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 800-

900bpPlasmid DNA sample

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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 No Insert

DNA gel

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Go to http://sequencing.retrogen.comOr: 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 Windowshttp://biologylabs.utah.edu/jorgensen/wayned/ape/

• pDRAW32– Windows onlyhttp://www.acaclone.com/