MSU & SkolTech Genetic engineering Sergiev P.V. 1755
Enzymes used for genetic engineering Restriction endonucleases 5' 3' 3' 5' 5'p 3' 5' 3' 3' 5' 3' p ' 5 recognition sites usually symmetrical ( ) EcoRI G AATTC SmaI CCC GGG PstI CTGCA G CTTAA G GGG CCC G ACGTG after cleavage phosphate is located at 5' side 1755
Enzymes used for genetic engineering DNA ligase 5'p 3' 5' 3' 3' 5' 3' p ' 5 T4 DNA ligase ATP , 5' 3' 3' 5' For ligation, phosphate should be at 5’-side 1755
Enzymes used for genetic engineering DNA polymerases 5' 3' 3' 5' DNA polymerase dATP,dCTP,dGTP,dTTP , 5' 3' 3' 5' 5' 3' 3' 5' DNA polymerase (exo) dATP,dCTP,dGTP,dTTP , 5' 3' 3' 5' DNA polymerases commonly used: Klenov fragment – medium polymerizing and 3’-exo activity T4 DNAP – strong polymerizing and 3’-exo activity Taq DNAP – thermostable polymerase, no 3’-exo activity (5’ exo) Pfu DNAP etc – thermostable polymerase, high 3’-exo activity 1755
Enzymes used for genetic engineering Reverse transcriptase (revertase, RT) 5' 3' 3' 5' RT dATP,dCTP,dGTP,dTTP , 5' 3' RT commonly used: AMV RT – medium processivity, RNaseH MuMLV RT – high processivity, RNase H various engineered RT for cDNA synthesis 1755
Enzymes used for genetic engineering RNA polymerase DNA Т7 promoter 5' 3' 3' 5' Т7 RNA polymerase ATP,CTP,GTP,UTP , 3' 5' RNA 1755
Enzymes used for genetic engineering Polynucleotide kinase (PNK) 5' 3' Т4 PNK ATP , 5'p 3' Alkaline phosphatase (AP) 5'p 3' AP 5' 3' 1755
Chemical synthesis of DNA Amidophosphite method NH N O B O B HO B O O O (MeO) 2 Tr N N (MeO) 2 Tr CF COOH O OMe O O 3 ... P ... O O B B O O (MeO) 2 Tr O O OMe ... P O B O N (MeO) 2 Tr OMe O P O O B O CH COOH, I , Py 3 2 O ... 1755
Chemical synthesis of DNA 96 at a time Typical oligonucleotide 16-20 nt synthesis for several hours Up to 120 nt 1755
DNA purification from cells Genomic DNA Phenol deproteinization Ethanol precipitation or binding to a column Plasmid DNA Binding to a column 1755
DNA amplificaton Polymerase chain reaction (PCR) 5' 3' 1X 5' 3' 96 o C, denaturation 5' 3' 5' 3' Specific temperature, hybridization 5' 3' 5' 3' 3' 5' 5' 3' 72 (or 68) o C, polymerization 5' 3' 5' 3' 2X 5' 3' 5' 3' 1755
DNA amplificaton Polymerase chain reaction (PCR) Taq DNA polymerase – “simple”, quick, cheap, but many errors Pfu and derivatives – slower, expensive but low error frequency RT PCR – reverse transcription, followed by PCR 1755
DNA amplificaton Quantitative PCR (qPCR) 1. Intercalating fluorophore SYBR green ( ) 2. Taqman - separation of fluorophore and quencher 1755
DNA (and RNA) sequencing Sanger sequencing 3' 5' 5' 3' ddA+dN C C G G G C G T G T G C A A T G C A T C A G C T G G C C C G C Ax G G C C C G C A C Ax G G C C C G C A C A C G T T Ax G G C C C G C A C A C G T T A C G T Ax G G C C C G C A C A C G T T A C G T A G T C G Ax O O O - O P O P O P O B O ddC+dN O - O - O - ddG+dN ddT+dN 1755
DNA sequencing Detection of the primer extension products ddA ddC ddG ddT T A G C 1. Radioactive labeling of the 5’-end G T A C 2. Radioactive labeling by a[32P]dNTP G T A T 3. Fluorescent labeling of the 5’-end G G T G 4. Fluorescent multcolor terminator T T nucleotides C C G A A C T G G 1755
Next generation sequencing Pyrosequencing (Roche 454) fragmentation conversion to blunt ends linker ligation purification on streptavidine beads 1755
Next generation sequencing Pyrosequencing (Roche 454) hybridization suspension PCR PCR mix streptavidin beads purification denaturation one bead - one type of DNA (but multiple copies!) 1755
Next generation sequencing Pyrosequencing (Roche 454) Sequential feeding by “ nano wells ” dATP, dCTP dGTP,dTTP , 400 000 wells detection of light emission dNTP dATP dCTP Ppi ATP свет sulfurylase, luciferase dGTP dTTP 1755
Next generation sequencing Pyrosequencing (Roche 454) 1 000 000 reads ~ 400 nt 10 hours ~ 1 000 000 000 nt/day Genome Sequencer FLX 1755
Next generation sequencing Illumina sequencing fragmentation conversion to blunt ends linker ligation PCR 1755
Next generation sequencing Illumina sequencing denaturation hybridization 1755
Next generation sequencing Illumina sequencing polymerization denaturation/hybridization 1755
Next generation sequencing Illumina sequencing polymerization denaturation restriction bridge PCR endonuclease 1755
Next generation sequencing Illumina sequencing DNA polymerase , fluorescent 3'-azidomethyl dNTP 1755
Next generation sequencing Illumina sequencing next scanning cycle TCEP demodification 1755
Applications of NGS Transcriptome (RNAseq) Genome wide binding sites of protein on DNA or RNA cross-linking, fragmentation purification sequencing 1755
Applications of NGS Translated regions (ribosome profiling) mRNA fragmentation, ultracentrifugation sequencing 1755
Applications of NGS chromosome conformation capture (3C) cross-linking, fragmentation ligation sequencing, analysis of hybrid reads 1755
Applications of NGS Identification of DNA, associated with specific compartment (DamID) Dam methylase, associated with specific nuclear compartment in vivo modification fragmentation purification sequencing 1755
Vectors Plasmid vector HindIII SphI Sse8387 PstI Usual features of BspMI SalI P-lac a plasmid vector SapI HincII AflIII AccI O-lac ori XbaI BamHI XmaI length up to 15 kbp KasI SmaI EheI AvaI NarI KpnI BbeI Asp718 ori lacZ SacI 2500 NdeI Ecl136 AlwNI BanII EcoRI selective marker ApoI 500 PUC19.TXT (antibiotic resistance) 2000 2686 bps AatII (regulated) promoter 1000 SspI 1500 polylinker AmpR Eam1105 XmnI BsaI Cfr10 ScaI GsuI 1755
Plasmid features Compatibility groups replication origins ColE1 (most frequently used) - pUC, pET, pQE etc 40-100 /cell p15A (rather frequent) pACYC - 10-12 /cell pSC 101 ( rare ) - pKD 1-2 /cell pCloDF13 exotic - pCDFDuet (Novagen) ( ) 20-40 /cell pRSF 1030 ( exotic pRSFDuet (Novagen) ) 100 /cell 1755
Plasmid features Antibiotic resistance Ampicillin (most frequent) Kanamycin (frequent) Chloramphenicol (occasionally used) Tetracyclin (occasionally used) Streptomycin (rare) Spectinomycin (rare) Zeocine (rare) Promoters lac (IPTG) taq (IPTG) lacUV5 (IPTG) T5lac (IPTG) T7 (needs T7 RNAP) T7lac (IPTG, needs T7 RNAP) ara (arabinose) tet (anhydroteracyclin) 1755
Vectors Phage vectors l M13 50 kbp up to , 23 kbp insert dsDNA used for cloning of large inserts 6 4 . kbp up to , 4 kbp insert ssDNA in virions dsDNA in cells , used for sequencing and mutagenesis 1755
Vectors Yeast vectors 2 m Centromere low copy number 1-3 high copy number 40-200 ARS + CEN ARS 2m URA3 URA3 HIS3 HIS3 LEU2 LEU2 TRP1 TRP1 ori ColE1 ori ColE1 AmpR AmpR 1755
Methods of bacterial transformation Chemical Electroporation CaCl 1.4 - 2.8 kV 2 Ultracompetent cells needs low conductivity o MnCl + growth at 18 C 2 + heat shock - + 1755
Methods for eukaryotes transformation + Yeast + + N + chemical transformation LiOAc + PEG ( ) + + - + O O - + + - - + - High eukaryotes : + - + - - cation lipid + + + electroporation + + + viruses + + microinjection oocytes ( ) + 1755
Heterologous protein expression Host selection E. coli P. pastoris mammalian S.cerevisiae baculovirus cells ++ + - ± ± yield ++ ++ ++ - -- simplicity - + ++ ++ ± Post translation modification - + ++ ++ ± folding 1755
Genome manipulation in bacteria Datsenko-Wanner method 5'-flank 3'-flank KanR PCR-product KanR 40 bp 40 bp electroporation l a b RED proteins - ( ) ' exonuclease ( ) ssDNA binding strand 5 , g annealing protein ( ) RecBCD inhibitor , gene of interst abg 1755
Genome manipulation in yeast Integration by homologous recombination marker 5'-flank 3'-flank >40 bp >40 bp yeast tarnsformation gene homologous recombination marker 1755
Methods for expression in eukaryotes Transient transfection plasmid vector expressed protein 48-72 hours 1755
Methods for expression in eukaryotes Stable cell line construction plasmid vector genes necessary viral particles for virus packaging with the gene of interest 1755
Methods for expression in eukaryotes Stable cell line construction lentivirus vector DNA intergated to the genome 1755
Inactivation of expression siRNA gene mRNA siRNA mRNA mRNA degradation mRNA 1755
Inactivation of expression shRNA gene shRNA mRNA mRNA mRNA degradation mRNA 1755
Transposase assisted knock-in Sleeping beauty system insert flanked by IR transposase expression vector 1755
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