Primer/probe design Crucial for successful DNA & RNA analysis! Main source of specificity for...

Primer/probe designCrucial for successful DNA & RNA analysis!• Main source of specificity for PCR

Primer/probe design• Also important for microarrays, sequencing, Southerns• Concerns•Specificity• Complementarity: •Hairpins• homoduplexes• heteroduplexes

may not meltMay be extended by DNA polymerase

Primer/probe design• Also important for microarrays, sequencing, Southerns• Concerns•Specificity• Complementarity: • Melting T• Should match!

Primer/probe design• Also important for microarrays, sequencing, Southerns• Concerns•Specificity• Complementarity: • Melting T• Should match!• Every site calculates them differently!

Primer/probe design• Also important for microarrays, sequencing, Southerns• Concerns•Specificity• Complementarity: • Melting T

• Targeting specific locations• amplifying specific sequences

• Targeting specific locations• amplifying specific sequences• creating mutations: need mismatch towards 5’ end so 3’ end binds well

• Targeting specific locations• amplifying specific sequences• creating mutations: need mismatch towards 5’ end so 3’ end binds well•Add restriction sites at 5’ end: may need to reamplify an amplicon

• Targeting specific locations• amplifying specific sequences• creating mutations: need mismatch towards 5’ end so 3’ end binds well•Add restriction sites at 5’ end: may need to reamplify an amplicon• Use Vent or another polymerase with proof-reading , taq’s error frequency is too high.

• Targeting specific locations• Amplifying sequences from related organisms• If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities

• Targeting specific locations• Amplifying sequences from related organisms• If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities• CodeHOP is a way around this: have a perfect match for 10-12 bases at 3’ end, then pick most likely candidates for the rest.

• Targeting specific locations• Amplifying sequences from related organisms• If use protein alignments need to make degenerate primers; eg CCN means proline, so need to make primers with all 4 possibilities• CodeHOP is a way around this: have a perfect match for 10-12 bases at 3’ end, then pick most likely candidates for the rest.•Based on codon usage

Primer/probe design• Stem-loop primers for short RNAs where only have enough info for one primer

Optimizing PCRChoosing enzyme• Template (RNA or DNA?)

Optimizing PCRChoosing enzyme• Template (RNA or DNA?)• Fidelity

Optimizing PCRChoosing enzyme• Template (RNA or DNA?)• Fidelity• Temperature stability

Optimizing PCRChoosing enzyme• Template (RNA or DNA?)• Fidelity• Temperature stability• Processivity

Optimizing PCRChoosing enzyme• Template (RNA or DNA?)• Fidelity• Temperature stability• Processivity• Km• dNTP• DNA

• Vmax

• Vmax• Tolerance of imperfect conditions•Dirty DNA•dNTP analogs or modified dNTP•[Mg] (or other divalent cation)

• Fragment ends

• Fragment ends• Cost

Optimizing PCRChoosing enzyme

• Template (RNA or DNA?): Reverse transcriptases from retroviruses make DNA copies of RNA•Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn2+

•Then dilute rxn & add Mg2+ to do PCR

• Template (RNA or DNA?)•Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn2+

•Then dilute rxn & add Mg 2+ to do PCR•Tfl DNA Polymerase from Thermus flavus has no RT activity: can mix with RNA & RT w/o activity then go directly to PCR after RT is done

Choosing enzyme• Template• Fidelity• Taq from Thermus aquaticus has no proof-reading

Choosing enzyme• Template• Fidelity• Taq from Thermus aquaticus has no proof-reading• goes faster, but error freq of 1 in 3000• Vent from Thermococcus litoralis has error frequency of 1 in 30,000

Choosing enzyme• Template• Fidelity• Taq from Thermus aquaticus has no proof-reading• goes faster, but error freq of 1 in 3000• Vent from Thermococcus litoralis has error frequency of 1 in 30,000• Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000

Choosing enzyme• Template• Fidelity• Taq from Thermus aquaticus has no proof-reading• goes faster, but error freq of 1 in 3000• Vent from Thermococcus litoralis has error frequency of 1 in 30,000• Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000•Genetically engineered proof-reading Phusion from NEB has error frequency of 1 in 2,000,000

Choosing enzyme• Template• Fidelity• Temperature stability• E.coli DNA polymerase I denatures at 75˚ C• T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C

Choosing enzyme• Template• Fidelity• Temperature stability• E.coli DNA polymerase I denatures at 75˚ C• T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C• T1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C

Choosing enzyme•Template• Fidelity• Temperature stability• E.coli DNA polymerase I denatures at 75˚ C• T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C• T1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C• T1/2 of Vent @ 95˚ C is 6.7 hours, 1.8 hours @ 100˚ C

Choosing enzyme•Template• Fidelity• Temperature stability• E.coli DNA polymerase I denatures at 75˚ C• T1/2 of Taq @ 95˚ C is 0.9 hours, < 0.1 hour @ 100˚ C• T1/2 of Phusion @ 96˚ C is >6 hours, 2 hours @ 98˚ C• T1/2 of Vent @ 95˚ C is 6.7 hours, 1.8 hours @ 100˚ C• T1/2 of Deep Vent from Pyrococcus species GB-D (grows @ 104˚ C)is 23 hours @ 95˚ C, 8 hours @ 100˚ C

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)• Q5 is 10x more processive than Pfu, 2x more than Taq

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)• Q5 is 10x more processive than Pfu, 2x more than Taq• lets you make longer amplicons in shorter time

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)• Q5 is 10x more processive than Pfu, 2x more than Taq• lets you make longer amplicons in shorter time• Taq = 8 kb max cf 40 kb for Q5

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km• dNTP: 13 µM for Taq, 60 µM for Vent

Choosing enzyme•Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km• dNTP: 13 µM for Taq, 60 µM for Vent• DNA: 2 nM for Taq, 0.01 nM for Deep Vent

Choosing enzyme•Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax: >1,000 nt/s when attached •Binding is limiting, processivity determines actual rate • 1000 bp/min is good for PCR

Choosing enzyme•Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax•Tolerance of imperfect conditions•Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax•Tolerance of imperfect conditions•Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion•dNTP analogs or modified dNTP•non-proofreading polymerases do better, but varies according to the modification

•[Mg]: Vent is more sensitive to [Mg] and needs 2x more than Taq

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax•Tolerance of imperfect conditions• Fragment ends: proof-readers (eg Vent) give blunt ends

Choosing enzyme• Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax•Tolerance of imperfect conditions• Fragment ends: proof-readers (eg Vent) give blunt ends• Non-proof-readers (eg Taq) give a mix of blunt & 3’A

Choosing enzyme•Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax•Tolerance of imperfect conditions• Fragment ends: proof-readers (eg Vent) give blunt ends• Non-proof-readers (eg Taq) give a mix of blunt & 3’A• Can use 3’A for t:A cloning

GAATTCAtcgcaCTTAAGtagcgt

Choosing enzyme•Template• Fidelity• Temperature stability• Processivity (how far does it go before falling off)•Km•Vmax•Tolerance of imperfect conditions• Fragment ends: proof-readers (eg Vent) give blunt ends• Cost @ NEB: http://www.neb.com/nebecomm/default.asp•Taq = $59.00 for 400 units•Vent = $62.00 for 200 units• Deep Vent = $90.00 for 200 units• Q5 = $ 103.00 for 100 units

Optimizing PCR• [enzyme] •[Template]• [Mg2+]• Annealing Temperature•Denaturation temperature

Optimizing PCR• [enzyme] • 0.4-2 units/100 µl for proofreaders : start with 1

Optimizing PCR• [enzyme] • 0.4-2 units/100 µl for proofreaders : start with 1• 1-5 units/100 µl for non-proofreaders : start with 3

Optimizing PCR• [enzyme] • [Template]• 1-10 ng/100 µl reaction for plasmids• 10 - 1000 ng/100 µl reaction for genomic DNA• Excess DNA can give extra bands, also brings more contaminants

• [dNTP]• 50-500 µM for Taq: start with 200, lower increases fidelity, higher increases yield• 200-400 µM for proof-readers: if too low start eating

Optimizing PCR• [enzyme] • [Template]• [Mg2+]• 0.5 - 4 mM for Taq: start with 1.5; lower if extra bands, raise if low yield

Optimizing PCR• [enzyme] • [Template]• [Mg2+]• 0.5 - 4 mM for Taq: start with 1.5; lower if extra bands, raise if low yield• 1- 8 mM for proofreaders: start with 2, lower if extra bands, raise if low yield

Optimizing PCR• [enzyme] • [Template]• [Mg2+]•Denaturation Temperature• Go as high as you can w/o killing enzyme before end• 94˚C for Taq• 96-98˚C for Vent• 98˚C for Deep Vent & Q5

Optimizing PCR• [enzyme] • [Template]• [Mg2+]•Denaturation Temperature• Annealing Temperature• Start 5 ˚C below lowest primer Tm

Optimizing PCR• [enzyme] • [Template]• [Mg2+]•Denaturation Temperature• Annealing Temperature• Start 5 ˚C below lowest primer Tm• Adjust up and down as needed

• # cycles: raise if no bands, lower if OK yield but extra bands

Optimizing PCR• Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles

Optimizing PCR• Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles• Can try “PCR enhancers” to overcome dirty DNA• Use Ammonium SO4 in buffer cf KCl• Use molecules that alter Tm eg DMSO & formamide• Use molecules that stabilise Taq eg Betaine & BSA

Optimizing PCR• Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles• If extra bands persist, use Taq bound to antibody•Inactive until denature antibody 7’ at 94˚ C

Optimizing PCR• Most common problems = wrong [DNA], dirty DNA, [Mg2+] annealing temperature & # cycles• If extra bands persist, use Taq bound to antibody•Inactive until denature antibody 7’ at 94˚ C•Alternatively, try touch-down: start annealing @ too high & lower 1˚ C each cycle ( binds correct target first)

Regulating transcription

Telling RNA pol to copy a DNA sequence

Transcription factors bind promoters & control initiation of transcription

1/signal gene senses

Regulating transcriptionTelling RNA pol to copy a DNA sequenceTranscription factors bind promoters & control initiation of transcription

1/signal gene senses1 binding site/signal gene senses

Transcription factorsBind surface -> base-pairs form unique patterns in major & minor grooves

Transcription factorsBind surface -> base-pairs form unique patterns in major & minor groovesScan DNA for correct pattern

Transcription factorsBind surface -> base-pairs form unique patterns in major & minor groovesScan DNA for correct patternneed 15 - 20 H-bonds = 5-8 base-pairs

Transcription

Prokaryotes have one RNA polymerase

makes all RNA

core polymerase = complex of 5 subunits (’)

Transcription

Prokaryotes have one RNA polymerase

makes all RNA

core polymerase = complex of 5 subunits (’)

not absolutely needed, but cells lacking are very sick

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters

Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters• Different sigmas bind different promoters

Primer/probe design Crucial for successful DNA & RNA analysis! Main source of specificity for...

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