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 • 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 2

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’ – 3’ – GCCAGGAGTGAAACGATG TCTAAAGGTGAAGAATTATTCACTGGTGTTGT

CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA

— 3’ — 5’

Denaturation Annealing

5’ – 3’ – GCCAGGAGTGAAACGATG — 3’

CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA

— 5’

Elongation

5’ – 3’ – GCCAGGAGTGAAACGATG

TCTAAAGGTGAAGAA

— 3’

CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA

— 5’ 4

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’ – 3’ –

TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC

ACTTACTTAACATGTTTGTGGTGGTGGTGGTG GTGATTCCGGTTCCACCG — 3’ — 5’

Denaturation Annealing

5’ –

TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC

3’ – GTGATTCCGGTTCCACCG — 3’ — 5’

Elongation

5’ –

TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC

3’ –

GTGGTGGTGGTGGTG

GTGATTCCGGTTCCACCG — 3’ — 5’ 5

Guidelines for Primer Design

• • • • • • • Length Melting Temperature – %GC Content Primer pair (length and T m ) 3’ GC Clamp Extra nucleotides for restriction enzymes No secondary structures Avoid cross-homology 6

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 10 bases 20 bases 30 bases 40 bases 50 bases % of Primers w/Correct Sequence (0.99) 10 (0.99) 20 (0.99) 30 (0.99) 40 (0.99) 50 = 90.4% = 81.8% = 74.0% = 66.9% = 60.5% 7

Guidelines: Melting Temperature (T

m

)

• • • • T m is the temperature at which 50% of the primer-target duplex dissociates Related to annealing temperature (T a ) for PCRs –

Aim for T a = 55-60°C, which corresponds to T m = 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% 8

Guidelines: Primer Pair

• • Forward and reverse primers should have similar length and T m – 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, T m = 53.5°C |Δlength| = 0 |Δtemperature| = 2.7°C Reverse Primer: 18nt, T m = 56.2°C 9

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 Restriction

Endonuclease

• Extra nucleotides for restriction enzyme (endonucleases) digestions DNA PDB: 1RVA 10

pZE_P

lac

-YPet

Sequencing primer will be provided for everyone seq_fwd 2886..2905

AmpR 2800..1940

7 BamHI (1) lac promoter 38..78

100 EcoRI (1) RBS 110..122

121 KpnI (1) pZE_Plac-YPET 3005 bp

General Workflow

1. Use pZE_P lac -Ypet as both your template DNA and as your plasmid backbone 2. Design primers to amplify Promoter and RBS region YPet 127..861

862 HindIII (1) T1 893..936

3. Use restriction enzyme (RE) sites (BamH1, EcoR1, Kpn1, or HindIII) Note: these sites are unique, double digest compatible and give ‘sticky ends’ colFWD T0 1827..1853

colE1 996..1803

lacI binding site seq_rev 1107..1088

colREV1 4. Create designed plasmid through ligation at RE sites colREV2 Seq FWD BamHI lac promoter -35 box -10 box EcoRI RBS KpnI YPET HindIII 11

Possible Cloning Strategies

RBS Modifications 1 BamHI lac promoter -35 box -10 box EcoRI RBS KpnI YPET HindIII 2 BamHI lac promoter -35 box -10 box EcoRI RBS KpnI YPET HindIII 12

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Possible Cloning Strategies

Promoter Modifications Changes in the -35 box lac promoter -35 box -10 box EcoRI RBS KpnI YPET BamHI HindIII 4 BamHI lac promoter -35 box -10 box BamHI EcoRI RBS KpnI Changes in sequence YPET EcoRI HindIII 13

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Possible Cloning Strategies

Other Methods QuickChange BamHI NNNNNN lac promoter -35 box -10 box NNNNNN EcoRI RBS KpnI YPET HindIII Error-Prone PCR 6 BamHI BamHI lac promoter -35 box -10 box EcoRI RBS KpnI YPET HindIII 14

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 800 900bp 16

• • • • •

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 17

But before you sequence…

• Verify plasmid DNA is plasmid DNA and contains insert of correct length – Colony PCR – – Restriction mapping DNA gel electrophoresis seq_fwd 2886..2905

+Insert No Insert AmpR 2800..1940

7 BamHI (1) lac promoter 38..78

100 EcoRI (1) RBS 110..122

121 KpnI (1) pZE_Plac-YPET 3005 bp YPet 127..861

862 HindIII (1) T1 893..936

seq_rev 1107..1088

T0 1827..1853

colE1 996..1803

DNA gel

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Go to http://sequencing.retrogen.com

Or: http://sequencing.laragen.com/ 19

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 24

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/ 25