2 تعریف:دست ورزی مستقیم ژنوم یک موجود با استفاده از فن آوری دی ان ای نوترکیباین فرایند شامل انتقال ماده ژنتیکی به موجود هدف می باشد که در این حالت ارگانیسم دریافت کننده، GMO (genetically modified organism) نامیده می شود.ZebrafischGloFish
3 مهندسی ژنتیک چیست؟ مهمترین بخش بیوتکنولوژی نوین شناسایی، جداسازی، بریدن وچسباندن مولکول های DNAایجاد DNA نوترکیببه طور کلی دستکاری ساختارهای ژنتیکی و خلق موجوداتی با ترکیب جدید ژنتیکی
4 تاریخچه 1970 شناسایی اولین آنزیم برشی توسط اسمیت Hind II)) 1971 تهیه اولین نقشه برشی DNA حلقوی ویروس SV40 با استفاده از آنزیم فوق1972 اولین تلاش ها برای ترکیب دو مولکول DNA از منشاء های مختلف توسط پائول برگ1973 ادغام DNA ویروسی و باکتریایی در یک پلاسمید توسط استنلی کوهن و هربرت بویر و تولید اولین موجود حامل DNA نوترکیب1977 تولید هورمون انسانی هوماتوستاتین توسط شرکت GenTech1977 ابداع روش خاتمه زنجیره برای توالی یابی DNA توسط Frederick Sanger
5 تاریخچه1978 تولید نخستین واکسن بر ضد هپاتیت B با استفاده از تکنیک DNA نوترکیب1983 ابداع روش واکنش زنجیره ای پلیمراز (PCR) توسط Kary Mullis1993 تولید هورمون رشد گاوی (BGH) در باکتری و خوراندن آن به گاوها برای افزایش تولید شیر آنها1994 تولید گوجه فرنگی فلاور ساور ( (FlavrSavr با عمر انبارداری طولانی تر1996 تولید بز تراریخت به نام گریس که دارای پروتئین ضد سرطان در شیر خود بود1997 تولد اولین موجود کلون شده به نام دالی در اسکاتلند1998 تا 2002 کلونینگ موجودات مختلف مثل مرغ، بز، میمون و گربه در اسکاتلند، کانادا و آمریکا
6 The most important topics: Gene Isolation & DNA extractionRestriction EnzymesDNA (Gene) cloningPCRPlasmids & vectorsGene transferHosts
7 DNA preparationThe procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages:A culture of bacteria is grown and then harvested.The cells are broken open to release their contents.This cell extract is treated to remove all components except the DNA.The resulting DNA solution is concentrated.
8 DNA Extraction1- Breaking the cell walls, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample.2- Removing membrane lipids by adding a detergent or surfactants.3- Removing proteins by adding a protease (optional but almost always done).4- Removing RNA by adding an RNase (often done).5- Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
12 Preparation of a cell extract The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid cell wall.with E. coli and related organisms, weakening of the cell wall is usually brought about by lysozyme, ethylene diamine tetra acetate (EDTA),or a combination of both.usually a detergent such as sodium dodecyl sulphate (SDS) is also added.
13 What does the detergent do? Each cell is surrounded by a cell membraneDNA is in the nucleus of a cell, which is also surrounded by a membrane
14 Detergent:Breaks apart membranes by attaching to the lipids (fats) & proteins in the membranes
16 Carbohydrates (sugars) DNA contaminants The cell and nuclear membranes have been broken apart, as well as all of the organelle membranes,such as those around the mitochondria and chloroplasts. So what is left?ProteinsRNACarbohydrates (sugars)DNAcontaminants
17 Purification of DNA from a cell extract Degradation of contaminantsSeparation of DNA from contaminants
18 Purification of DNA from a cell extract The standard way to deproteinize a cell extract is to add phenol or a 1 : 1 mixture of phenol and chloroform. These organic solvents precipitate proteins but leave the nucleic acids (DNA and RNA) in aqueous solution. In some case treat the cell extract with a protease such as pronase or proteinase K before phenol extraction. Some RNA molecules, especially messenger RNA (mRNA), are removed by phenol treatment, but most remain with the DNA in the aqueous layer. The only effective way to remove the RNA is with the enzyme ribonuclease, which rapidly degrades these molecules into ribonucleotide subunits.
20 Using ion-exchange chromatography to purify DNA from a cell extract
21 Concentration of DNA samples Collecting DNA by ethanol precipitation.(b) For less concentrated solutions ethanol is added (at a ratio of 2.5 volumes of absolute ethanol to 1 volume of DNA solution) and precipitated DNA collected by centrifugation.(a) Absolute ethanol is layered on top of a concentrated solution of DNA. Fibers of DNA can be withdrawn with a glass rod.
22 Other methods for the preparation of total cell DNA )The CTAB method( Plant tissues are particularly difficult in this respect as they often contain large amounts of carbohydrates that are not removed by phenol extraction. One method makes use of a detergent called cetyl trimethyl ammonium bromide (CTAB), which forms an insoluble complex with nucleic acids. When CTAB is added to a plant cell extract the nucleic acid–CTAB complex precipitates, leaving carbohydrate, protein, and other contaminants in the supernatant. The precipitate is then collected by centrifugation and resuspended in 1 M sodium chloride, which causes the complex to break down. The nucleic acids can now be concentrated by ethanol precipitation and the RNA removed by ribonuclease treatment.
23 Preparation of plasmid DNA separation of plasmid DNA from the large amount of bacterial chromosomal DNASeparation on the basis of sizeSeparation on the basis of conformation
26 Ethidium bromide–caesium chloride density gradient centrifugation Purification of plasmid DNA by EtBr–CsCl density gradient centrifugation
27 Gel Electrophoresis DNA is placed at one end of a gel A current is applied to the gelDNA molecules are negatively charged and move toward positive end of gelSmaller molecules move faster than larger ones
39 Manipulation of Purified DNA Almost all DNA manipulative techniques make use of purified enzymesWithin the cell these enzymes participate in essential processes such as DNA replication and transcription,breakdown of unwanted or foreign DNA (e.g., invading virus DNA), repair of mutated DNA, and recombination between different DNA molecules
40 The range of DNA manipulative enzymes DNA manipulative enzymes can be grouped into four broad classes, depending on the type of reaction that they catalyze:Nucleasesare enzymes that cut, shorten, or degrade nucleic acid moleculesLigasesjoin nucleic acid molecules togetherPolymerasesmake copies of moleculesModifying enzymesremove or add chemical groups
41 NucleasesNucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.There are two different kinds of nuclease:Exonucleasesremove nucleotides one at a time from the end of a DNA molecule.Endonucleasesare able to break internal phosphodiester bonds within a DNA molecule.
45 Two type of endonuclease (a) S1 nuclease, which cleaves only single- stranded DNA, including single-stranded nicks in mainly double-stranded molecules.(b) DNase I,which cleaves both single- anddouble-stranded DNA.
46 Ligases(a) Repair of a discontinuity—a missing phosphodiester bond in one strand of a double-stranded molecule.(b) Joining two molecules together.
47 PolymerasesDNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template(a) The basic reaction: a new DNA strand is synthesized in the 5′ to 3′ direction.
48 DNA polymerase I(b) DNA polymerase I, which initially fills in nicks but then continues to synthesize a new strand, degrading the existing one as it proceeds.
49 The Klenow fragment(c) The Klenow fragment, which only fills in nicks.
50 Reverse transcriptase The ability of this enzyme to synthesize a DNA strand complementary to an RNA template is central to the technique called complementary DNA (cDNA) cloning.(d) Reverse transcriptase, which uses a template of RNA.
51 The Taq DNA polymeraseThe Taq DNA polymerase used in the polymerase chain reaction (PCR) is the DNA polymerase I enzyme of the bacterium Thermus aquaticus.
52 DNA modifying enzymesThere are numerous enzymes that modify DNA molecules by addition or removal of specific chemical groups.The most important are as follows:Alkaline phosphatasePolynucleotide kinaseTerminal deoxynucleotidyl transferase
53 Alkaline phosphataseAlkaline phosphatase (from E. coli, calf intestinal tissue, or arctic shrimp), which removes the phosphate group present at the 5′ terminus of a DNA molecule.
54 Polynucleotide kinase Polynucleotide kinase (from E. coli infected with T4 phage), which has the reverse effect to alkaline phosphatase, adding phosphate groups onto free 5′ termini.
55 Terminal deoxynucleotidyl transferase Terminal deoxynucleotidyl transferase (from calf thymus tissue), which adds one or more deoxyribonucleotides onto the 3′ terminus of a DNA molecule.
56 Restriction endonucleases Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion.
57 The discovery and function of restriction endonucleases The initial observation that led to the eventual discovery of restriction endonucleases was made in the early 1950s, when it was shown that some strains of bacteria are immune to bacteriophage infection, a phenomenon referred to as host- controlled restriction.over 2500 different ones have been isolated and more than 300 are available for use in the laboratory.
58 The function of a restriction endonuclease in a bacterial cell (a) phage DNA is cleaved, but (b) bacterial DNA is not.
59 Classification of restriction endonuclease Three different classes of restriction endonuclease are recognized:Types I and III are rather complex and have only a limited role in genetic engineering.Type II restriction endonucleases, on the other hand, are the cutting enzymes that are so important in gene cloning.
60 The recognition sequences for some of the most frequently used restriction endonucleases
62 Isoschizomers ; Neoschizomers; Isocaudomers Restriction enzymes that are isolated from different organisms and recognize identical sequences are called isoschizomers.NeoschizomersIsoschizomeric enzymes that possess different cleavage sites in a particular recognition sequence are called neoschizomers(e.g., Sma I [CCC↓GGG] and XmaC I [C↓CCGGG]).Isocaudomersare pairs of restriction enzymes that have slightly different recognition sequences but upon cleavage generate identical termini. For example the enzymes Mbo I and BamH I are isocaudomers:
63 IsocaudomersThe same sticky ends produced by different restriction endonucleases
64 The frequency of recognition sequences in a DNA molecule The number of recognition sequences for a particular restriction endonuclease in a DNA molecule of known length can be calculated mathematically.A tetranucleotide sequence(e.g., GATC) should occur once every 44 = 256 nucleotides, and a hexanucleotide (e.g., GGATCC) once every 46 = nucleotides.These calculations assume thatthe nucleotides are ordered in a random fashionfour different nucleotides are present in equal proportions (i.e., the GC content = 50%).
65 Ligation – joining DNA molecules together The final step in construction of a recombinant DNA molecule is the joining togetherof the vector molecule and the DNA to be cloned
66 Sticky ends increase the efficiency of ligation Ligation of complementary sticky ends is much more efficient.This is because compatible sticky ends can base pair with one another by hydrogen bonding forming a relatively stable structure for the enzyme to work on.Putting sticky ends onto a blunt-ended molecule:LinkersAdaptorsProducing sticky ends by homopolymer tailing
72 Producing sticky ends by homopolymer tailing Tailing involves using the enzyme terminal deoxynucleotidyl transferase to add a series of nucleotides onto the 3′-OH termini of a double-stranded DNA molecule.If this reaction is carried out in the presence of just one deoxyribonucleotide, a homopolymer tail is produced.
73 homopolymer tailingFrequently polydeoxycytosine (poly(dC)) tails are attached to the vector and poly(dG) to the DNA to be cloned.
74 Repair of the recombinant DNA molecule In practice, the poly(dG) and poly(dC) tails are not usually exactly the same length, and the base-paired recombinant molecules that result have nicks as well as discontinuities.Repair is therefore a two-step process:Using Klenow polymerase to fill in the nicksUsing DNA ligase to synthesize the final phosphodiester bonds.
78 Horizontal Gene transfer in bacteria There are three processes by which exogenous genetic material may be introduced into a bacterial cell:Transformation(Capturing DNA from solution)Transduction(Phage-mediated)Conjugation(Bacterial Sex)
79 transformationIn molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s).
80 Transformation was first demonstrated in 1928 by British bacteriologist Frederick Griffith. Frederick Griffith (1879 - 1941) British physician, pathologist, bacteriologist Known for discovery of pneumococcal transformation
83 Avery, MacLeod and McCarty, in 1944 discovered that DNA is the material of which genes and chromosomes are made.Oswald Theodore Avery ForMemRS (October 21, 1877 – February 2, 1955) was a Canadian-born American physician and medical researcher.Maclyn McCarty (South Bend, Indiana, June 9, – January 2, 2005) was an American geneticist.Colin Munro MacLeod (January 28, 1909 — February 11, 1972) was a Canadian-American geneticist.
84 Avery–MacLeod–McCarty experiment Chemical analysis showed that the proportions of carbon, hydrogen, nitrogen, and phosphorus in this active portion were consistent with the chemical composition of DNA. To show that it was DNA rather than some small amount of RNA, protein, or some other cell component that was responsible for transformation, Avery and his colleagues used a number of biochemical tests. They found that trypsin, chymotrypsin and ribonuclease (enzymes that break apart proteins or RNA) did not affect it, but an enzyme preparation of "deoxyribonucleodepolymerase" (a crude preparation, obtainable from a number of animal sources, that could break down DNA) destroyed the extract's transforming power
85 The Hershey–Chase experiments in 1952 helped to confirm that DNA was the genetic material. Martha Cowles Chase (November 30, 1927 – August 8, 2003 was an American geneticist and Alfred Hershey’s laboratory assistantAlfred Day Hershey (December 4, 1908 – May 22, 1997) was an American Nobel Prize-winning bacteriologist and geneticist.
86 Hershey–Chase experiment Hershey and Chase were testing two competing hypotheses:1- DNA is the genetic material2- protein is the genetic materialDNA was radiolabeled with radioactive phosphorus-32 (32P)Protein was radiolabeled with radioactive sulfur-35 (35S)
87 Hershey–Chase experiment radioactive sulfur-35 (35S) tracer found in the supernatant with the phage coatsDNAis genetic material
88 Transformation:uptake of naked DNA (DNA without associated cells or proteins) by competent bacteriacompetent :competence is the ability of a cell to take up extracellular ("naked") DNA from its environment.natural competenceinduced or artificial competence
89 Artificial competence Use of divalent cationsmost commonly calcium chloride (CaCl2) solution under cold condition, and then exposed to a pulse of heat shock.ElectroporationIn this method the cells are briefly shocked with an electric field of kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.
90 Polymerase Chain Reaction (PCR) A scientific technique in molecular biology to amplify a single or a few copies of a piece of DNADeveloped in 1983 by Kary MullisKary Mullis wined Nobel Prize in Chemistry in 1993Kary Banks Mullis(born December 28,1944) Nobel Prize winning American biochemist.
91 PCR - Applications DNA cloning Forensic medicine In-vitro mutation DNA sequencingGene identificationMolecular phylogeneticsDetection of disease mutationsGene expression studies22
98 Number of short products synthesized after 25 cycles of PCR with different numbers of starting molecule.Note: The numbers assume that amplification is 100% efficient, none of the reactants becoming limiting during the course of the PCR.
99 PCR componentsA basic PCR set up requires several components and reagents. These components include:DNA template Two primers: reverse & forwardTaq DNA polymerase* dNTPs (Deoxynucleoside triphosphates : dATP, dGTP, dCTP, dTTP)Buffer solutionMgCl2 (Divalent cations, magnesium or manganese ions; generally Mg2+ is used,)dd water* Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965.
103 annealing temperature The ideal annealing temperature must be :low enough to enable hybridization between primer and templatehigh enough to prevent mismatched hybrids from formingThis temperature can be estimated by determining the melting temperature or Tm of the primer–template hybrid.The Tm is the temperature at which the correctly base-paired hybrid dissociates (“melts”). A temperature 1–2°C below this should be low enough to allow the correct primer–template hybrid to form, but too high for a hybrid with a single mismatch to be stable. The Tm can be determined experimentally but is more usually calculated from the simple formula:Tm = (4 × [G + C]) + (2 × [A + T])°C
107 PCR - Disadvantages Need sequence information Limited amplification sizeLimited amounts of productInfidelity of DNA replication21
108 The error rate of Taq polymerase The error rate has been estimated at one mistake for every 9000 nucleotides of DNA that is synthesized, which might appear to be almost insignificant but which translates to one error in every 300 bp for the PCR products obtained after 30 cycles.
110 Nested PCRNested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment.The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.The advantage of nested PCR is that if the wrong PCR fragment was amplified, the probability is quite low that the region would be amplified a second time by the second set of primers. Thus, Nested PCR is a very specific PCR amplification.
115 Real time pcr TaqMan® Probes Molecular Beacons Scorpions® FRET Probes SYBR® Green AssayHigh Resolution Melting Analysis (HRMA)Dual Labeled Probe:TaqMan® ProbesMolecular BeaconsScorpions®FRET Probes
116 High Resolution Melting (HRM) MechanismHigh Resolution Melting Analysis (HRM) is a post PCR method. The region of interest within the DNA sequence is first amplified using the polymerase chain reaction. During this process, special saturation dyes are added to the reaction, that fluoresce only in the presence of double stranded DNA. Such dyes are known as Intercalating dyes.During PCR, the region of interest amplified is known as Amplicon. As the amplicon concentration in the reaction tube increases the fluorescence exhibited by the double stranded amplified product also increases.After the PCR process the HRM analysis begins. In this process the amplicon DNA is heated gradually from around 50°C up to around 95°C. As the temperature increases, at a point the melting temperature of the amplicon is reached and the sample DNA denatures and the double stranded DNA melts apart. Due to this the fluorescence fades away.This is because in the absence of double stranded DNA the intercalating dyes have nothing to bind to and they only fluoresce at a low level. This observation is plotted showing the level of fluorescence vs the temperature, generating a Melting Curve. Even a single base change in the sample DNA sequence causes differences in the HRM curve. Since different genetic sequences melt at slightly different rates, they can be viewed, compared, and detected using these curves.
118 Advantages of HRMCost Effective High Resolution DNA Melt Analysis is cost effective than other genotyping technologies such as sequencing and Taqman SNP typing. This makes it ideal for large scale genotyping projects.Simple and Fast Work flow After performing PCR amplification of the target sequence, no additional instrumentation is required for HRM Analysis.Fast and Powerful HRM Analysis is fast and powerful, due to which it is able to accurately genotype huge number of samples in a short time, with a high level of accuracy.Low Reagent Consumption High Resolution Melting Analysis requires only PCR reaction volume for analysis of each sample, eliminating the need for HPLC solvents or Denaturing Gradient Gel Electrophoresis (DGGE) gels.
119 Applications of HRM SNP Genotyping DNA Mapping Mutation Scanning Species IdentificationZygosity testingDNA Methylation AnalysisDNA Fingerprinting
120 TaqMan® probe functioning While carrying out a TaqMan® experiment, a fluorogenic probe, complementary to the target sequence is added to the PCR reaction mixture. This probe is an oligonucleotide with a reporter dye attached to the 5' end and a quencher dye attached to the 3' end. Till the time the probe is not hydrolized, the quencher and the fluorophore remain in proximity to each other, separated only by the length of the probe. This proximity however, does not completely quench the flourescence of the reporter dye and a background flourescence is observed. During PCR, the probe anneals specifically between the forward and reverse primer to an internal region of the PCR product. The polymerase then carries out the extension of the primer and replicates the template to which the TaqMan® is bound. The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter molecule away from the close vicinity of the quencher. The fluorescence intensity of the reporter dye, as a result increases. This process repeats in every cycle and does not interfere with the accumulation of PCR product.
122 TaqMan® probe applications: 1. Quantitative real time PCR. 2. DNA copy number measurements. 3. Bacterial identification assays. 4. SNP genotyping. 5. Verification of microarray results.
123 Molecular BeaconsIntroduction to Molecular Beacons Molecular beacons are single stranded hairpin shaped oligonucleotide probes. In the presence of the target sequence, they unfold, bind and fluoresce. The molecular beacon chemistry is one of the chemistries used to carry out a real time experiment.
124 Molecular Beacons Structure A molecular beacon consists of 4 parts, namely:Loop:This is the base pair region of the molecular beaconwhich is complementary to the target sequence.Stem:The beacon stem sequence lies on both the ends of the loop.It is typically 5-7 bp long at the sequences at both the ends arecomplementary to each other.5' fluorophore:Towards the 3' end of the molecular beacon, is attached a dyethat fluoresces in presence of a complementary target.3' quencher (non fluorescent):The quencher dye is covalently attached to the 3' end of the molecular beacon and when the beacon is in closed loop shape, prevents the fluorophore from emitting light.
125 Applications of Molecular Beacons SNP detectionReal-time nucleic acid detectionReal-time PCR quantificationAllelic discrimination and identificationMultiplex PCR assaysDiagnostic clinical assays
126 Scorpion primers & probes Structure of the Scorpion primerThe Scorpion primer carries a Scorpion probe element at the 5' end. The probe is a self- complementary stem sequence with a fluorophore at one end and a quencher at the other. The Scorpion primer sequence is modified at the 5'end. It contains a PCR blocker at the start of the hairpin loop (Usually HEG monomers are added as blocking agent). In the initial PCR cycles, the primer hybridizes to the target and extension occurs due to the action of polymerase. Scorpion primers can be used to examine and identify point mutations by using multiple probes. Each probe can be tagged with a different fluorophore to produce different colors.Scorpion® Primer functioningIn Scorpion primers, the probe is physically coupled to the primer which means that the reaction leading to signal generation is a unimolecular one. This is in contrast to the bi- molecular collisions required by other technologies such as TaqMan® or Molecular Beacons. After one cycle of PCR extension completes, the newly synthesized target region will be attached to the same strand as the probe. Following the second cycle of denaturation and annealing, the probe and the target hybridize. The denaturation of the hairpin loop requires less energy than the new DNA duplex produced. Consequently, the hairpin sequence hybridizes to a part of the newly produced PCR product. This results in the separation of the fluorophore from the quencher and causes emission.
130 Fluorescence resonance energy transfer (FRET) FRET probes are a pair of fluorescent probes placed in close proximity. Fluorophores are so chosen that the emission spectrum of one overlaps significantly with the excitation spectrum of the other. During FRET, the donor fluorophore excited by a light source, transfers its energy to an acceptor fluorophore when positioned in the direct vicinity of the former.The acceptor fluorophore emits light of a longer wavelength, which is detected inspecific channels. The light source cannot excite the acceptor dye.FRET probes functioningThe hybridization probe system consists of two oligonucleotides labeled with fluorescent dyes. The hybridization probe pair is designed to hybridize to adjacent regions on the target DNA.Each probe is labeled with a different marker dye. Interaction of the two dyes can only occur when both are bound to their target. The donor probe is labeled with fluorophore at the 3' end and the acceptor probe at 5' end. During PCR, the two different oligonucleotides hybridize to adjacent regions of the target DNA such that the fluorophores, which are coupled to the oligonucleotides, are in close proximity in the hybrid structure. The donor fluorophore (F1) is excited by an external light source, then passes part of its excitation energy to the adjacent acceptor fluorophore (F2). The excited acceptor fluorophore (F2) emits light at a different wavelength which can then be detected and measured.
137 vectorsa vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell.The four major types of vectors are: plasmids can hold up to 10 kbviruses (Viral vectors)Cosmids can hold up to 30 kb artificial chromosomes (BAC, YAC) bacs are able to hold up to 350 kb of DNA
140 plasmidsa plasmid is a double-stranded DNA molecule, in many cases, circularPlasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre ring in Saccharomyces cerevisiae).Plasmid sizes: vary from 1 to over 1,000 kbp.Copy number: in a single cell can range anywhere from one to thousands under some circumstances.Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.a plasmid can contain inserts of only about 1–10 kbp
141 Plasmid typesAnother way to classify plasmids is by function. There are five main classes:Fertility F-plasmidswhich contain tra genes. They are capable of conjugation.Resistance (R)plasmidswhich contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Historically known as R-factors, before the nature of plasmids was understood.Col plasmidswhich contain genes that code for bacteriocins, proteins that can kill other bacteria.Degradative plasmidswhich enable the digestion of unusual substances, e.g. toluene and salicylic acid.Virulence plasmidswhich turn the bacterium into a pathogen.Plasmids can belong to more than one of these functional groups.
142 Plasmid DNA may appear in one of five conformations in a gel: Nicked open-circular DNA has one strand cut.Relaxed circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed (supercoils removed). This can be modeled by letting a twisted extension cord unwind and relax and then plugging it into itself.Linear DNA has free ends, either because both strands have been cut or because the DNA was linear in vivo. This can be modeled with an electrical extension cord that is not plugged into itself.Supercoiled (or covalently closed-circular) DNA is fully intact with both strands uncut, and with an integral twist, resulting in a compact form. This can be modeled by twisting an extension cord and then plugging it into itself.Supercoiled denatured DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.slowfast
144 Plasmids containing: Origin of replication Multiple cloning site (MCS, or polylinker)which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this locationSelectable marker geneA selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection.Selectable markers are often antibiotic resistance genes
145 Replication strategies for (a) a non-integrative plasmid, and (b) an episome.
149 Incompatibility groups Several different kinds of plasmid may be found in a single cell. In fact, cells of E. coli have been known to contain up to seven different plasmids at once.incompatibility is due to the sharing of one or more elements of the plasmid replication or partitioning systemsIncompatibility may be:symmetric(either coresident plasmid is lost with equal probability)vectorial(one plasmid is lost exclusively or with higher probability than the other)
150 Some cloning vectors TA Cloning Vectors Bluescribe puc pJET pBR322
151 Plasmid featuresSizePlasmids range from about 1.0 kb for the smallest to over 250 kb for the largest plasmidscopy numberThe factors that control copy number are not well understood. Some plasmids, especially the larger ones, are stringent and have a low copy number of perhaps just one or two per cell; others, called relaxed plasmids, are present in multiple copies of 50 or more per cell.
152 Viral vectorsDelivery of genes by a virus is termed transduction and the infected cells are described as transduced.
153 Some other viral vectors use mostly in gene therapy & vaccination: Lambda phageM13 bacteriophageSome other viral vectors use mostly in gene therapy & vaccination:Retroviruses (including lentiviruses) – integrating mammalianviruses; useful for stable/long-term gene expressionAdeno-associated virus – replication-deficient parvoviruses, canintegrate into a host cell genome; perspective vectors for geneTherapyAdenovirus – non-integrating DNA-genome virus; largeapplication area from research to gene vaccination and anti-cancerAgentsPoxvirus – large cytoplasmic DNA virus; perspective fordevelopment of gene vaccination
155 Lambda phageLambda phage is a head & tail virus, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibersUsually, a "lytic cycle" ensues, where the lambda DNA is replicated many times and the genes for head, tail and lysis proteins are expressed. This leads to assembly of multiple new phage particles within the cell and subsequent cell lysis, releasing the cell contents, including virions that have been assembled, into the environment. However, under certain conditions the phage DNA may integrate itself into the host cell chromosome in the lysogenic pathway.
162 M13 bacteriophageM13 is a filamentous bacteriophage composed of circular single stranded DNA (ssDNA) which is 6407 nucleotides long encapsulated in approximately 2700 copies of the major coat protein P8, and capped with 5 copies of two different minor coat proteins (P9, P6, P3) on the ends. The minor coat protein P3 attaches to the receptor at the tip of the F pilus of the host Escherichia coli. Infection with filamentous phages is not lethal; however, the infection causes turbid plaques in E. coli. It is a non-lytic virus. However a decrease in the rate of cell growth is seen in the infected cells. M13 plasmids are used for many recombinant DNA processes, and the virus has also been studied for its uses in nanostructures and nanotechnology.
163 M13 Replication in E. coliBelow are steps involved with replication of M13 in E. coli.Viral (+) strand DNA enters cytoplasmComplementary (-) strand is synthesized by bacterial enzymesDNA Gyrase, a type II topoisomerase, acts on double-stranded DNA and catalyzes formation of negative supercoils in double-stranded DNAFinal product is parental replicative form (RF) DNAA phage protein, pII, nicks the (+) strand in the RF3'-hydroxyl acts as a primer in the creation of new viral strandpII circulizes displaced viral (+) strand DNAPool of progeny double-stranded RF molecules producedNegative strand of RF is template of transcriptionmRNAs are translated into the phage proteinsPhage proteins in the cytoplasm are pII, pX, and pV, and they are part of the replication process of DNA. The other phage proteins are synthesized and inserted into the cytoplasmic or outer membranes.pV dimers bind newly synthesized single-stranded DNA and prevent conversion to RF DNARF DNA synthesis continues and amount of pV reaches critical concentrationDNA replication switches to synthesis of single-stranded (+) viral DNApV-DNA structures from about 800 nm long and 8 nm in diamterpV-DNA complex is substrate in phage assembly reaction
165 cosmidA cosmid is a plasmid that contains a cos site from the lambda genome.
166 Cosmid vectorsThe vector replicates as a plasmid (it contains a ColE1 origin of replication), uses Ampr for positive selection and employs lambda phage packaging to select for recombinant plasmids carrying foreign DNA inserts 45 KB in size.Ligation of cosmid vector and foreign DNA fragments (SauIIIA partial digest fragments 45 kb in size) is similar to ligation into a lambda substitution vector. The desired ligation product is a concatemer of 45 kb foreign DNA fragment and 5 kb cosmid vector sequences.
167 cosmidThis concatemer is then packaged into viral particles (remember packaging is cos site to cos site) and these are used to infect E. coli where the cosmid vector replicates using the ColE1 orignin of replication. Phage packaging serves only to select for recombinant molecules and to transfer these long DNA molecues (50 kb total) into the bacterial host (50 kb fragments transform very inefficiently while phage infection is very efficient).
168 YAC yeast artificial chromosome YAC vector is capable of carrying a large DNA fragment (up to 2 Mb), but its transformation efficiency is very low.
169 YAC Essential components of YAC vectors Procedure Centromers (CEN), Telomeres (TEL)Autonomous replicating sequence (ARS) for proliferation in the host cell.Ampr for selective amplificationMarkers such as TRP1 and URA3 for identifying cells containing the YAC vector.Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)Procedure1- The target DNA is partially digested by EcoRI and the YAC vector is cleaved by EcoRI and BamHI.2- Ligate the cleaved vector segments with a digested DNA fragment to form an artificial chromosome.3- Transform yeast cells to make a large number of copies.
171 BAC Bacterial artificial chromosome Bacterial Artificial Chromosomes (BAC) have been developed to hold much larger pieces of DNA than a plasmid can (They are able to hold up to 350 kb of DNA).BAC vectors were originally created from part of an F’ plasmid. In 1992, Hiroaki Shizuya took the parts of F’ that were important, cleaned it up, and turned it into a vector.With these vectors it is possible to study larger genes, several genes at once, or entire viral genomes.
172 Common gene components oriS, repE – Ffor plasmid replication and regulation of copy number.parA and parBfor partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC.A selectable markerantibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection.T7 & Sp6phage promoters for transcription of inserted genes.
174 Some of the scientific practices that have simplified with BAC vectors includes: BACs are often used to sequence the genome of organisms in genome projects, for example the Human Genome Project.phylogenetic studieswhat the absolute minimum size of a genome could be.BAC vectors are also useful for studying pathogens,helpful in the development of vaccines.BAC vectors are playing a tremendous role in discovering new and powerful antibiotics in the environment.Discovering enzymes that are able to help clean up oil spillshelp farmers breed healthier farm animalsprocess radioactive waste
177 Expression vectorAn expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin that are important for medical treatments of specific diseases like diabetes.
178 Expression vector features PromoterShine-Dalgarno / kozak sequenceStart codonTags and fusion proteinsProtease cleavage siteStop codonTranscription terminatorRegulatory gene (repressor)
180 Regulatory gene (repressor) Many promoters show leakiness in their expression i.e. gene products are expressed at low level before the addition of the inducer. This becomes a problem when the gene product is toxic for the host. This can be prevented by the constitutive expression of a repressor protein.
181 PromoterThe promotor initiates transcription and is positioned nucleotides upstream of the ribosome binding site.The ideal promoter exhibits several desirable features:It is strong enough to allow product accumulation up to 50% of the total cellular protein.It has a low basal expression level (i.e. it is tightly regulated to prevent product toxicity).It is easy to induce.
182 Shine-Dalgarno sequence The Shine-Dalgarno (SD) sequence is required for translation initiation and is complementary to the 3'-end of the 16S ribosomal RNA. The efficiency of translation initiation at the start codon depends on the actual sequence. The concensus sequence is: 5'-TAAGGAGG-3'. It is positioned 4-14 nucleotides upstream the start codon with the optimal spacing being 8 nucleotides.The Kozak consensus sequenceKozak consensus or Kozak sequence, is a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G‘. The Kozak consensus sequence plays a major role in the initiation of the translation process. The sequence was named after its discoverer, Marilyn Kozak.
183 The Shine-Dalgarno sequence (or Shine-Dalgarno box), proposed by Australian scientists John Shine (b.1946) and Lynn Dalgarno (b.1935),Marilyn Kozak, PhD, professor of biochemistry at UMDNJ-Robert Wood Johnson Medical School.Lynn DalgarnoMarilyn KozakJohn Shine
184 Tags and fusion proteins N- or C-terminal fusions of heterologous proteins to short peptides (tags) or to other proteins (fusion partners) offer several potential advantages:Improved expressionFusion of the N-terminus of a heterologous protein to the C-terminus of a highly- expressed fusion partner often results in high level expression of the fusion protein.Improved solubilityFusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.Improved detectionFusion of a protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody or a binding protein (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) allows for detection of a protein during expression and purification.Improved purificationSimple purification schemes have been developed for proteins fused at either end to tags or proteins which bind specifically to affinity resins
185 Protease cleavage site Protease cleavage sites are often added to be able to remove a tag or fusion partner from the fusion protein after expression. However, cleavage is rarely complete and often additional purification steps are required.Start codonInitiation point of translation. In E. coli the most used start codon is ATG. GTG is used in 8% of the cases. TTG and TAA are hardly used.Stop codonTermination of translation. There are 3 possible stop codons but TAA is preferred because it is less prone to read-through than TAG and TGA. The efficiency of termination is increased by using 2 or 3 stop codons in series.Transcription terminatorThe transcription terminator reduces unwanted transcription and increases plasmid and mRNA stability.
187 Use of linkers , adaptors & technique of homopolymer tailing Can we made sticky end from blunt end DNA?Use of linkers , adaptors & technique of homopolymer tailing
188 Linkers: short pieces of double-stranded DNA synthesized in the test tubeIt is blunt-endedcontains a restriction siteDNA ligase can attach linkers to the ends of target DNA moleculeslinkers, can be made in very large amounts and added into the ligation mixture at a high concentration.More than one linker will attach to each end of the DNA molecule
190 Adaptors: There is one potential drawback with the use of linkers. Target DNA may containing same restriction endonuclease recognition sequence/ sequences.How we can avoid of this problem?Adaptors,Like linkers, are short synthetic oligonucleotides. But unlike linkers,an adaptor is synthesized so that it already has one sticky end.
193 technique of homopolymer tailing A homopolymer is simply a polymer in which all the subunits are the same.Tailing involves using the enzyme terminal deoxynucleotidyl transferase to add a series of nucleotides onto the 3'-OH termini of a double stranded DNA molecule.If this reaction is carried out in the presence of just one deoxyribonucleotide a homopolymer tail is produced.In practice the poly( dG) and poly( dC) tails are not usually exactly the same length.Repair is therefore a two step process, using Klenow polymerase to fillin the nicks followed by DNA ligase to synthesize the final phosphodiester bonds.
196 If the complementary homopolymer tails are longer than about 20 nucleotides, is often stable enough to be introduced into the host cell directly without repair . Once inside the host, the cell’s own DNA polymerase and DNA ligase repair the recombinant DNA molecule, completing the construction begun in the test tube.
197 T4 DNA LigaseCatalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1). Applications:Cloning of restriction fragmentsJoining linkers and adapters to blunt-ended DNA
201 Alkaline PhosphataseAlkaline Phosphatase catalyzes the removal of 5´ phosphate groups from DNA, RNA, ribo- and deoxyribonucleoside triphosphates. Since CIP-treated fragments lack the 5´ phosphoryl termini required by ligases, they cannot self-ligate (1). This property can be used to decrease the vector background in cloning strategies. Source: Calf intestinal mucosa (CIP) or Shrimp (SAP)Applications:Removing 5´ phosphates from DNA, RNA, rNTPs and dNTPsPreparation of templates for 5´end labelingPrevention of recircularization of cloning vectorsDephosphorylation of serine, threonine and tyrosine residues in protein
203 Types of librariesThe DNA cloned in libraries depends on the purposes of the investigatorsGenomic library: A collection of fragments representing all DNA in the chromosomes of a cell. Genomic libraries are made from total nuclear DNA of an organism.Chromosome-specific libraries: are made from the DNA of purified isolated chromosomes.cDNA library: Collection of DNAs representing the genes expressed in cell or tissue type. cDNA libraries are made from the mRNA populations of particular tissues. There are thus liver, heart, leaf, root, etc. cDNA libraries.The advantage of cDNA library is that it contains only the coding region of a genomecDNA expression libraries: A collection of expressed genes cloned into a vector next to a regulatory region so that the protein will be produced under conditions controlled by the experimenter. For example, DNA can be cloned into a vector to create a fusion protein used to test a gene product for interaction with other proteins.Electronic libraries: With the human genome project, the DNA sequence from several model organisms as well as humans is being determined. This information serves as a virtual DNA library that can be analyzed via computer
206 DIGESTION OF GENOMIC DNA Complete digestion with an endonuclease will result in a library containing no overlapping fragments:Complete digestion has these restriction:1- produce many of fragment that construction of library and screening is difficult2- If a sequence has more than 1 recognision site for an enzyme, caused cloning of fragment in form of 2 or more unoverlapping fragments. In this condition, we need walking clones to findout overlapping fragments3- Because of nonrandomly disribution of restriction sites on DNA, such a sequence may located on a fragment which so very large or small that can’t be cloned.Solving of problems:1- mechanical shearing: hasn’t desirable efficiency2- partial digestion: main method
207 PARTIAL DIGESTION Adventages: Incomplete Digestion of Genomic DNA will allow identification of sequence overlapsHowever, incomplete digestion will result in a library containing overlapping fragments: Use the reduction of enzyme concentration or time of activityAdventages:Thus, the sequence information obtained from one clone will allow the isolation of clones containing neighboring (overlapping) sequence information.This can allow large contiguous stretches of sequence information to be obtained ("Chromosome Walking"). overlapping fragments allows larger physical maps to be constructed as contiguous chromosomal regions (contigs) are put together from the sequence dataReduction the size of library (large size of fragment): number of clones needed to fully represent the genomeWithout information abuot the location of restriction sites near the tag sequence , it can be isolate the fragments
208 a “contig” Partial restriction enzyme digestion allows cloning of overlapping fragmentsa “contig”isolation of ~20 kb fragments provides optimallysized DNAs for cloning in bacteriophagepartial digestion with a frequent-cutter (4-base cutter) allows productionof overlapping fragments, since not every site is cutoverlapping fragments insures that all sequences in the genome are cloned
209 All possible sites:Results of a partial digestion:= uncut= cut
210 Restriction endonucleases Restriction enzymes cut DNA into specific fragmentsFrequency of cutting (if frequency of all bases are equall)4-base cutter 44 = bp5-base cutter 45 = 1,024 bp6-base cutter 46 = 4,096 bp8-base cutter 48 = 65,536 bpIn practice, the actual number of fragments is quite different because the distributionof nucleotides is non-random and most organismes do not have an equal number ofthe four basesfor example; human DNA has overall only 40% G + C and the frequency of the dinucleotide CpG is only 20% of that expected. In addition many cytosine residues are methylatedand this can prevent restriction endonucleases digetion.E.g.: Not I (8-base cutter 48 = 65,536 bp or 65.5 Kb) but produce fragmentswith average size of Kb
211 cuts DNA into 256 bp average-sized fragments in a random sequence 4-base cutter:cuts DNA into 256 bp average-sized fragments in a random sequenceevery 256 bp: NO256 bp average-size fragments: YESBar = 256 b
212 Construct of cDNA library Procedure: 1- Total RNA extraction (tRNA, rRNA, mRNA, snRNA,… ) 2- Isolation of mRNA from Total RNA 3- Synthesis of sscDNA from mRNA 4- produce of dscDNA from mRNA 5-Insertion of dscDNA in to the cloning vectors 6- Transformation (transduction) in to the host cells
215 cDNA library construction: Replacement synthesis of second strand of cDNA 5’AAAAA3’ mRNA(all mRNAs in cell)anneal oligo(dT) primers of bases in length5’AAAAATTTTT3’5’3’add reverse transcriptase and dNTPs5’3’AAAAATTTTT3’5’ cDNAadd RNaseH (specific for the RNA strand of an RNA-DNAhybrid) and carry out a partial digestion5’3’AATTTTTshort RNA fragments serve as primers forsecond strand synthesis using DNA polymerase I
216 5’ 3’ AAAAA TTTTT AAA TTTTT 5’ 3’ AAA TTTTT 5’ 3’ 5’ 3’ AAAAA TTTTT short RNA fragments serve as primers forsecond strand synthesis using DNA polymerase IAAATTTTT5’3’DNA polymerase I removes the remaining RNA withits 5’ to 3’ exonuclease activity and continues synthesisAAATTTTT5’3’DNA ligase seals the gaps5’3’AAAAATTTTTdouble-strandedcDNA
217 5’ 3’ AAAAA TTTTT AAAAANNNNNNNNG TTTTTNNNNNNNNCTTAA 5’ 3’ EcoRI linkers are ligated to both endsusing DNA ligaseAAAAANNNNNNNNGTTTTTNNNNNNNNCTTAA5’3’AATTCNNNNNNNNGNNNNNNNNdouble-stranded cDNA copies of mRNA with EcoRI cohesive ends arenow ready to ligate into a bacteriophage lambda vector cut with EcoRI
230 Transient vs. Stable Transient transfection In transient transfection, the transfected DNA is not integrated into host chromosome. DNA is transferred into a recipient cell in order to obtain a temporary but high level of expression of the target gene.Stable transfectionStable transfection is also called permanent transfection. By the stable transfection, the transferredDNA is integrated (inserted) into chromosomal DNA and the genetics of recipient cells is permanent changed.
231 Transfection Methods Generally, there are 9 ways for gene transfer: (1) Lipid-mediated method;(2) Calcium-phosphate mediated;(3) DEAE-dextran-mediated;(4) Electroporation;(5) Biolistics;(6) Viral vectors;(7) Polybrene;(8) Laser transfection;(9) Gene transfection enhanced by elevated temperature (Sambrook, 2001).
232 Indirect gene transfer (Vector-mediated) Direct gene transfer (Vectorless)
233 Direct gene transfer (Vectorless) Chemical methodsElectroporationBiolistic, Particle gun deliveryLipofectionMicroinjection and macroinjectionLaser inducedFibremediated
234 Chemical mediated gene transfer Chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts. Calcium phosphate is also used to transfer DNA into cultured cells.
235 Microinjection where the DNA is directly injected into protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped ( micrometer diameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
236 Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA. The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
240 liposomesA vesicle is a small bubble enclosed by lipid bilayer. Vesicles can form naturally, for example, during the endocytosis, or they may be prepared.Artificially prepared vesicles are known as liposomes.
241 LipofectionLipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid to form an aggregate with the negatively charged (anionic) genetic material. A net positive charge on this aggregrate has been assumed to increase the effectiveness of transfection through the negatively charged phospholipid bilayer.
242 Lipofection advantages high efficiencyability to transfect all types of nucleic acids in a wide range of cell typesease of usereproducibilitylow toxicityIn addition, this method is suitable for all transfection applications (transient, stable, co-transfection, reverse, sequential or multiple transfections…). High throughput screening assay and has also shown good efficiency in some in vivo models.
247 Gene transfer methods Vectorless or direct gene transfer Chemical mediated gene transfer(polyethylene glycol(PEG))Microinjection / MacroinjectionElectroporationParticle gun/Particle bombardment (gene gun)Liposome mediated gene transfer or LipofectionVector-mediated or indirect gene transferAgrobacterium as gene transfer systemViral mediated gene transfer
248 DIRECT GENE TRANSFER Chemical methods Electroporation Particle gun LipofectionMicroinjection And macroinjectionPollen transformation Delivery Via growing pollen tubesLaser inducedFiber mediated
249 Agrobacterium tumefaciens Agrobacterium tumefaciens (updated scientific name: Rhizobium radiobacter) is the causal agent of crown gall disease (the formation of tumours) in over 140 species of dicot. It is a rod shaped, Gram negative soil bacterium.A. tumefaciens is an alphaproteobacterium of the family Rhizobiaceae, which includes the nitrogen fixing legume symbionts. Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are pathogenic and do not benefit the plant.
250 Tumour-inducing plasmid (Ti plasmid( a tumour-inducing plasmid (Ti plasmid or pTi), of 200 kb, which contains the T-DNA and all the genes necessary to transfer it to the plant cell. ( Many strains of A. tumefaciens do not contain a pTi.)
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