Primer/probe design Crucial 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 melt May 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

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well

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

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

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

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T 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 bases at 3’ end, then pick most likely candidates for the rest.

Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T 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 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 PCR Choosing enzyme Template (RNA or DNA?)

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation)

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation) Fragment ends

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Fidelity Temperature stability Processivity Km dNTP DNA Vmax Tolerance of imperfect conditions Dirty DNA dNTP analogs or modified dNTP [Mg] (or other divalent cation) Fragment ends Cost

Optimizing PCR Choosing 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 Mn 2+

Optimizing PCR Choosing 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 Mn 2+ Then dilute rxn & add Mg 2+ to do PCR

Optimizing PCR Choosing enzyme Template (RNA or DNA?) Tth DNA Polymerase from Thermus thermophilus reverse transcribes RNA in the presence of Mn 2+ 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 T 1/2 of 95˚ C is 0.9 hours, < ˚ C

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of 95˚ C is 0.9 hours, < ˚ C T 1/2 of 96˚ C is >6 hours, 2 98˚ C

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of 95˚ C is 0.9 hours, < ˚ C T 1/2 of 96˚ C is >6 hours, 2 98˚ C T 1/2 of 95˚ C is 6.7 hours, ˚ C

Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of 95˚ C is 0.9 hours, < ˚ C T 1/2 of 96˚ C is >6 hours, 2 98˚ C T 1/2 of 95˚ C is 6.7 hours, ˚ C T 1/2 of Deep Vent from Pyrococcus species GB-D 104˚ C)is 23 95˚ C, 8 100˚ 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

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 GAATTCAtcgca CTTAAGtagcgt

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 NEB: Taq = $59.00 for 400 units Vent = $62.00 for 200 units Deep Vent = $90.00 for 200 units Q5 = $ for 100 units

Optimizing PCR [enzyme] [Template] [Mg 2+ ] Annealing Temperature Denaturation temperature

Optimizing PCR [enzyme] units/100 µl for proofreaders : start with 1

Optimizing PCR [enzyme] units/100 µl for proofreaders : start with units/100 µl for non-proofreaders : start with 3

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

Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants [dNTP] µM for Taq: start with 200, lower increases fidelity, higher increases yield µM for proof-readers: if too low start eating

Optimizing PCR [enzyme] [Template] [Mg 2+ ] mM for Taq: start with 1.5; lower if extra bands, raise if low yield

Optimizing PCR [enzyme] [Template] [Mg 2+ ] 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] [Mg 2+ ] 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] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm

Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed

Optimizing PCR [enzyme] [Template] [Mg 2+ ] 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, [Mg 2+ ] annealing temperature & # cycles

Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles Can try “PCR enhancers” to overcome dirty DNA Use Ammonium SO 4 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, [Mg 2+ ] 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, [Mg 2+ ] 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 too high & lower 1˚ C each cycle ( binds correct target first)

Regulating transcription Telling RNA pol to copy a DNA sequence

Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiation of transcription

Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiation of transcription 1/signal gene senses

Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiation of transcription 1/signal gene senses 1 binding site/signal gene senses

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

Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves Scan DNA for correct pattern

Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves Scan DNA for correct pattern need 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 Prokaryotes 1) Core RNA polymerase is promiscuous

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters

Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters Different sigmas bind different promoters