Generating the histone codeCDK8 kinases histones to repress transcription

Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcription

Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genes

Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!

Generating the histone codeCDK8 kinases histones to repress transcriptionAppears to interact with mediator to block transcriptionPhosphorylation of Histone H3 correlates with activation of heat shock genes!Phosphatases reset the genes

Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcription

Generating the histone codeRad6 proteins ubiquitinate histone H2B to repress transcriptionPolycomb proteins ubiquitinate histone H2A to silence genesA TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing

Generating the histone codeMany proteins methylate histones: highly regulated!

Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activity

Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!

Generating the histone codeMany proteins methylate histones: highly regulated!Methylation status determines gene activityMutants (eg Curly leaf) are unhappy!Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow)& H3K9me3 (red)

Generating the histone codeChromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)Histone demethylases have been recently discovered

Next step: deciding which genes to clone•Problem = correlating enzymes with genes• Who matches the pH?• Who localizes where?• Which isoform if alternatively spliced?

Clone several, using one known to work to find orthologsUse sequence to design primers to clone cDNA

How to proceed?

Kinetic and Spectroscopic Studies of Bicupin OxalateOxidase and Putative Active Site Mutants

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

•good primers only bind your sequence

Primer/probe design• Also important for microarrays, sequencing, Southerns• Concerns

•Specificity: only want them to bind at one place•Main concern: 3’ end should not bind

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!• 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• creating mutations: need mismatch towards 5’ end so 3’ end binds well

•Add restriction sites at 5’ end: may need to reamplify an amplicon

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• 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)

• Phusion 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 Phusion

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

•Vmax: >1,000 nt/s when attached •Binding is limiting, processivity determines actual rate • 1000 bp/min is good for PCR

•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• 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• Phusion = $ 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

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 & Phusion

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

• Use temperature gradient feature to find best T

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)