V21 DNA Cloning PDF Lecture Notes

V21: DNA Cloning Lecture BIO 111 / 117 – Molecular and classical genetics Lecture 21: DNA cloning...

V21: DNA Cloning Lecture BIO 111 / 117 – Molecular and classical genetics Lecture 21: DNA cloning [email protected] Hot spring in Yellowstone National Park, USA – the home 1 of Thermus aquaticus, the source of Taq DNA polymerase. Overview BIO 111 / 117 part II - Molecular Genetics (lecture # in blue) 25 Mutations DNA repair 26 The dynamic genome P13 P 14 Central dogma of gene expression P in bacteria P 19 P 15 Regulation in eukaryotes 20 P 17 Genetic code processing P 16 Applications DNA cloning 21 P18 Protein synthesis Modern recombinant DNA techniques Genomics and 23 the human genome 25 2  Repetition Gene Regulation General Aspects What we learned so far… 3 Genregulation in Eukaryoten Repetition Gene Regulation Prokaryotes The lac operon of E. coli kann hydrolisieren ↳ alolactose herstellung The lac operon contains three structural genes, coding respectively for b-galactosidase ↳ kann (lacZ), lactose permease (lacY), and a third lactose-metabolizing enzyme (thiogalactoside induziert werden transacetylase, lacA). der Moleküle vorhanden immer ein paar Kopien ! When lactose is NOT present, only a few molecules of each protein are present in the cell. When lactose is present (and glucose is not), the rate of synthesis of the three enzymes increases ~ 1000 x within 2-3 minutes. This substrate-dependent increase in enzyme synthesis is known as induction. durch Genexpression s strukturgene reguret D ()).. 4 S EGalaclosidase ↓ B- ↳Lactoseese 3. Strukturgen wichtig) promotor Laktose (nicht so => lactose => in die Zelle hydrolistert V19: The lac Operon Prokaryotes lac operon in the absence of lactose repressor, /exprimiert In the absence of lactose, the lac repressor LacI (encoded by the lacI gene, which is NOT part of the lac operon) binds to the lac operator (lacO). The lacO operator overlaps with the lacP promoter. Binding of the LacI repressor to the operator prevents transcription of the operon by RNA polymerase. O + > - bindet wenn an keine polymerase operator lactose kann vorhanden mit an I -O promotor binden keine ) expression = des operons 5 V19: The lac Operon Prokaryotes lac operon in the absence of lactose When lactose is present, b-galactosidase converts some of it into allo-lactose. ↳ isomerisierung-- Allo-lactose acts as an inducer for the lac operon: allo-lactose can bind to the LacI repressor, and induce a allosteric (conformational) change. LacI that has bound kannanebinden O wird allo-lactose looses its peroxprimient & glactosidase/ parmease/ ability to bind to the Claktose - verwertung) lacO operator. RNA polymerase is now free O kann nicht meur to transcribe the an operator binden operon. bindet an > - Strukturver- repressor änderung & inducer 6  Repetition Gene Regulation Prokaryotes Positive/negative regulation vs. inducible/repressible operons Positive vs. negative regulation: What effect does binding of the regulatory protein to the operator have? – Promotes gene expression à positive regulation, activator protein erhört Genexpression > - – Inhibits gene expression à negative regulation, repressor protein inhibiert Genexpression > - Inducer ausgeschalten substrate lines synthesweges Es ↑ normalzustand: Inducible vs. repressible operons: What effect does binding of the small molecule to the regulatory protein have? - normally not expressed – Promotes gene expression à inducible operon, molecule is an inducer – Inhibits gene expression à repressible operon, molecule is a co-repressor ↓ ↓ normally expressed produkt eines Synthese - weges 7 => Genexpression eines operous wird inhibiert Repetition Gene Regulation Prokaryotes Positive/negative regulation vs. inducible/repressible operons Positive inducible operons: activator protein + inducer Positive repressible operons: activator protein + co-repressor Negative inducible operons: repressor protein + inducer ↳ z B lac. operon Negative repressible operons: repressor protein + co-repressor 8  Repetition Gene Regulation Prokaryotes Constitutive and uninducible lac operon mutations immer angeschalten In constitutive mutants, the lac operon is In uninducible mutations, the lac operon transcribed even in the absence of is non-functional, even in the presence of lactose. lactose. Two types of constitutive mutants were Four types of uninducible mutants were kann nicht operator mehr an identified: binden identified: a I induzierbar - – Mutations in lacI (lacI ) repressor > - verändert – Mutations in lacZ (lacZ-) phänotyp nicht , aber Transkription – Mutations in lacO (lacOc) mutation im operator – Mutations in lacY (lacY-) > - kann nicht binden nicht binden ↳ repressor – Mutations in lacP (lacP-) polymerase kann > - polymerase kann immer binden => – Mutations in lacI (lacIs) bindet > - immer - an operator auch wenn es inducer transkribieren ↳ super repressor gibt => phânotyp : uninducible Question: both lacI- and lacOc mutations have the same phenotype (constitutive expression). Similarly, lacZ-, lacY-, lacP-, and lacIs all have the same phenotype (uninducible). Suggest three different experiments that would allow to you distinguish 9 between mutations in each group. Repetition Gene Regulation Prokaryotes Transcriptional regulation of the lac operon - summary a. In the absence of lactose, the LacI repressor - - sits on the lacO operator and prevents RNA polymerase from transcribing the lac operon. => wenige Kopien - Very very little lac mRNA vonpermeasdas b. In the presence of both glucose and lactose, LacI does not bind lacO. The affinity of RNA - polymerase for the lacP promoter is low, and only a few mRNAs are transcribed. c. In the presence of lactose and absence of glucose, LacI does not bind lacO, and the CAP protein (activator ( -bindet an cAMP*CAP complex binds upstream of the stark ↳ bindet vor promotor lacP promoter, and enhances recruitment of RNA exprimient => sehr starke induzierte Gehexpress. des lac operons polymerase. The lac operon is efficiently transcribed. & lactose lac operon nicht viel glucose => 10 stark exprimiert  Repetition Gene Regulation Eukaryotes cis-acting regulatory sequences in eukaryotic genes (lecture 15) There are three major classes of cis-regulatory sequences in eukaryotic protein-coding genes: – Promoters, which bind RNA polymerase II – Promoter-proximal elements (upstream > - stromaufwärts - wie stark promoter elements, UPEs), which bind proteins promotor : zum könnenweitein wird ein Gen kriptionsstart sein that assist binding of RNA pol II. UPEs are exprimiert? located 100-200 bp upstream of the O transcription start site. – => Enhancers and silencers, which bind proteins wo a wann wird ein that regulate gene expression through several Gen exprimiert? possible mechanisms. - Proteins that bind to eukaryotic regulatory I sequences are generally known as transcription promotor : Kann DNA polymerase factors. binden 11 Repetition Gene Regulation Eukaryotes Enhancers and silencers Enhancers and silencers are regulatory elements that can control the activity of a promoter even at a great distance (up to 100 kb!). Enhancers/silencers can be located either upstream or downstream of the promoter (in the gene, downstream of the gene, pretty much wherever). Enhancers increase transcription rates. Gregulatorische bestimmen wann & wo ein Gen - Netzwerke wird Silencers reduce transcription. exprimiert Enhancers and silencers are usually responsible for directing the expression of a gene to certain cell types or certain developmental periods. weit enhancer : relativ weg 000 00 O kb stromaufwärts dpp Stromabwärts - Some of the enhancers elements controlling expression of the Drosophila dpp gene Question: Enhancers can work on promoters that are very far away. How do enhancer-binding 12 proteins know which promoter to activate? Why don’t they activate all promoters they are close to?  DNA frei räumen Offen : von Histone > damit DWA polymerase binder - - kann => Chromatin Repetition remodeling Gene Regulation Eukaryotes The problem with chromatin -geschlossen Eukaryotic DNA is tightly packaged into Eukaryotic transcription factors will thus chromatin. use several different mechanisms to promote gene transcription: How can RNA polymerase II even – Direct interaction with RNA pol II access the promoter and unwind the double helix? – Indirect interaction with RNA pol II (via coactivators) – Chromatin remodeling – Modulation of chromatin condensation Bestimmung der Zugänglichkeit ↳ Kondensationsstufe 13 Repetition Gene Regulation Eukaryotes Chromatin can exist at different levels of packaging (condensation) Eukaryotic chromatin can exist in various states of condensation, which are detectable by microscopy. Euchromatin: open chromatin. Genes in euchromatin regions can be transcribed (if the right transcription factors are present). Gene zugänglich können exprimiert , werden Heterochromatin: condensed chromatin. Genes in heterochromatin regions are usually silenced (not expressed). in Proteinen verpackt unzugänglich für > - transcription factors & INA polymerase – Constitutive heterochromatin: always condensed. ↳ Centromere ↳ offnet fast wie – Facultative heterochromatin: Regions that can be open under some conditions (e.g. in some cell types, or during some developmental stages), and condensed under others. 14 manchmal offen , sonst geschlossen/Kondensiert  Repetition Gene Regulation Eukaryotes Chromatin remodeling protein can nudge nucleosomes von nucleosome Even in open chromatin, a promoter might ↓ verdeckt be inaccessible to RNA pol II if the TATA box - happens to be tightly bound to a nucleosome. Chromatin remodeling complexes have the- capacity to displace individual nucleosomes. RNA I kann polymerase ⑪- binden ↓ This can move the TATA box into a free DNA transcription region, where it can then be bound by TBP. ↓ richtigen tata box binding protein zur -stelle 15 Repetition Gene Regulation Eukaryotes How is chromatin condensation regulated? Modification of core histones histore code Methylation of DNA Stable association with specific proteins or RNAs 16  Im nucleosome > - 8 histone > - histore tails Repetition Gene Regulation Eukaryotes Acetylation of histone tails Acetylation occurs on lysines residues, and is sequence specific. Acetylation of histones results in the opening of the nucleosomal structure (possibly through charge neutralization). – Acetylated histones = active chromatin – Deacetylated histones = inactive chromatin Acetylation is reversible Chromatin aktiv > – Acetylation: Histone acetyl-transferase (HAT) - As Lysine aus ↳ inaktiv – Deacetylation: Histone deacetylase (HDAc) > Chromatin - Dopor E HAT phosphates O * -- + t - [ Deacytelation - --- HDAC 17 histone tails histone tails nicht mehr an DNA binden sehr stark gebunden DNA-Stränge an => DNA offen & nucleosome kann transkribiert => DNA kann nicht werden Repetition Trauskribertwerdese ist Gene Regulation Eukaryotes Heterochromatin DNA is often methylated on C In mammals and plants, Cytosine residues in a CG dimer sequence are often methylated on C5 in heterochromatic regions. 5’ … mC G… 3’ HC - 3’ … G mC… 5’ O 4 mini indrom 5 3 The 5-methyl modification of C does not affect its base 6 2 1 pairing with G, but can be recognized by proteins that bind to methylated DNA and recruit HDAcs, thereby ~ entfernen insuring continued repression of gene expression. Acetylierungen histone tails binden an DNA => DNA nicht 5-methylcytosine zugänglich für transcription factors => Genexpression repressed 18  Repetition Gene Regulation Eukaryotes Epigenetic inheritance nur durch modifikation der proteine/ DNA = nicht mutationen methylierung der Heritable modification in gene function / gene expression that is not due to changes in the base sequence in the DNA. Epigenetic states can be inherited through mitosis, and in some cases even through meiosis (i.e., from one generation to the next). Epigenetic inheritance can be due to stable association of protein with a region of DNA, or the stable modification of this DNA (e.g. methylation). Genome + Environment = Phenotype Nature Nurture 19 Repetition Gene Regulation Eukaryotes X chromosome inactivation During early embryonic development, each cell in a female embryo randomly inactivates (“shuts down”) one of its two X chromosomes. The inactivated chromosome becomes heterochromatin (visible as the Barr body in microscopy). The X chromosome inactivation pattern is stably inherited to all daughter cells of that embryonic cell: the inactivated chromosome stays inactivated, the active chromosome stays activated. Mammalian females are thus functionally mosaics: they have some patches of cells that express genes from only one X chromosome, and other patches that express genes only from the other chromosome. Question: If what I say above is true, then why are X-linked mutations recessive in women? Should not 50% of the cells in heterozygous carriers actually be “mutant”? 20  Repetition Gene Regulation Eukaryotes Parental imprinting IGF2 ↑ IGF 2 R (Rezeptor) Some autosomal genes (e.g., igf2, which codes for a - growth factor) are systematically silenced (by mothers) in the maternal germline, such that only the paternal copy is expressed in the progeny. Urkeimzelle Other genes (e.g. receptor for IGF2) show the opposite phenotype and are inactivated in males. Silencing of inactive genes is likely mediated through DNA methylation (and chromatin modification?). aktiv In the germ line, all imprinting marks are removed from geschlechtspezifisch both parental copies, and the proper “gender pattern” is Optinaktiv 0 0 put on both chromosomes (which will be transmitted with this “gender pattern” to the next generation). 21 Repetition Gene Regulation Eukaryotes Gene expression can be regulated at many different levels Regulation of gene expression is cool! D I - ↓ miRNAs Protein- Phospholierung/ Steller in der Zelle 22 V21: DNA Cloning Lecture BIO 111 / 117 – Molecular and classical genetics Lecture 21: DNA cloning [email protected] Hot spring in Yellowstone National Park, USA – the home 23 of Thermus aquaticus, the source of Taq DNA polymerase. After this lecture you will be able to answer / explain How restriction enzymes work. How to generate recombinant DNA molecules. DNA cloning. What a cloning vectors is. How DNA gel electrophoresis works. How polymerase chain reaction (PCR) works. 24 V21: Recombinant DNA Technology General Aspects Recombinant DNA technology Recombinant DNA technology = Set of molecular tools used for locating, isolating, - altering, amplifying or studying DNA segments. - - Genetic engineering = application of recombinant DNA technology to solve practical ( problems in biology, medicine, veterinary medicine, agriculture, and other areas. Synthetic biology = Synthetic biology is the engineering of biology: the synthesis of complex, biologically based (or inspired) systems, which display functions that do not exist in nature. 25 V21: Recombinant DNA Technology General Aspects Design and synthesis of a minimal bacterial genome was ist nötig für Leben ? Mycoplasma mycoides (JCV-syn3.0) 473 genes, 531 kb genome 26  Glucose Aufnahme V21: Recombinant Blutzuckert DNA Technology Example of genetic engineering: General Aspects Tackling diabetes type 1 & 2 Solution: Insulin Source: wikipedia ~9 million people with T1D ~100 million people with T2D need insulin (that’s about 72,000 kg insulin) gankeine Insulin herstellung 27 V21: Recombinant DNA Technology Example of genetic engineering: General Aspects production of human insulin Before 1980s: type I diabetics must take insulin isolated from pancreas of slaughtered animals (pigs). hohe expression Idea: express the human insulin gene in bacteria (same genetic code!) at high levels, - purify the protein, give it to patients instead of the animal insulin. - Advantages – No immune reaction (human protein) – Pure protein (no contaminants) – No risk of transfer of animal diseases (viruses, prions) – Cheap! - gentechnisch modifizierbar => bessere Insulinmoleküle 28 https://www.nature.com/articles/s41586-024-08042-3 V21: Recombinant DNA Technology General Aspects The many hurdles faced by recombinant DNA 1. Genes are minute, and there are thousands of them in every cell. How can you find the one gene that you care about? 2. Even if you found your gene, how would you isolate it from the rest of the genome? 3. Even if you could isolate it from the rest of the genome, how would you put it into bacteria? 4. Even if you could put it into bacteria, how would you identify the rare bacterium that did take up the DNA? 5. Even if you could identify the rare bacterium that took up the DNA, how would you insure that the human gene would be replicated during cell division? 6. Even if you could make it replicate, how would you make sure that the human gene would be transcribed and translated properly? 29 V21: DNA Cloning General Aspects How can we make many pure copies of a specific DNA molecule? 30 V21: DNA Cloning PCR How to amplify a DNA fragment of interest insulin The problem: genes are minute, and there are thousands of them in every cell. How do clowing you get enough DNA of the gene you care PCR about to actually be able to work with it? Den sanian 0 O -flanking primers Solution: selective amplification of your favorite gene (often abbreviated yfg-1) your favorite gene in Vector einfügen /amplification fin Two general approaches for amplifying (making many copies) of a DNA molecule: a. Cloning b. Polymerase chain reaction (PCR) ampliene 31 Methode zur & Gewinnung identischen Vervielfältigung V21: DNA Cloning eines Stücks DNA aus Donor in einer Wirtszelle General Aspects Cloning (molecular biology) Cloning: Selective manipulation followed by amplification of a donor DNA molecule of interest in a host cell or organism. Donor: Cell or organism from which we wish to amplify a DNA fragment. Host: Cell or organism into which we will introduce the donor DNA in order to amplify it. 32 V21: DNA Cloning General Aspects Can human genes (or human chromosomes) get replicated on their own in bacteria? No ! because : E coli cannot the enkaryotic orgins of. recognize replication DNA- > Universell 33 - orgin of replication wird benötigt V21: DNA Cloning Vectors Plasmids Plasmids are small, circular extrachromosomal DNA molecules present in many bacterial species. oft zusätzliche Gehe die nützlich sind In the wild, plasmids often carry genes that are not essential for bacterial function, but that can play important roles in the life cycle or growth of bacteria: – Mating – Metabolism of unusual molecules – Resistance to antibiotics Plasmids range in size from a few kb to a few hundred kb. Plasmids have their own origin of replication, and replicate independently of the bacterial chromosome. O Stringent plasmids are present in only one or two copies per cell, relaxed plasmids 34 Lcan be present in many (up to many dozen) copies. sehr wenig Kopien 1-2  V21: DNA Cloning Vectors Cloning vectors A cloning vector is a stable, self-replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into and replication in a host cell. An effective vector has three important characteristics: (bakteriell) 1. An origin of replication, which allows replication within ori the host cell. 2. One or more selectable markers, which allows the selective growth or identification of cells that have Marker ampicivin) taken up the vector. (z B Antibiotikaresistenz. - > 3. One or more unique sites, into which the foreign DNA schnittstellen DNA can be inserted. ↓ einzigartige Enzymstellen no man schneiden kann Most common bacterial cloning vectors are modified plasmids. 35 V21: DNA Cloning Vectors Plasmid vectors origin of replication O Modern plasmid vectors are very sophisticated derivatives of the selection original wild plasmids. ↳marken Most modern plasmids carry: – A selection marker (e.g., a resistance gene for ampicillin) – A “polylinker” or “multiple cloning site” (MCS) containing unique restriction sites for several different enzymes Promotor las operon – An inducible bacterial promoter upstream of the MCS. O ori 36 V21: DNA Cloning Vectors How can you get target molecules “into” cloning vectors? - Restriction enzymes Recombination enzymes (not covered in this lecture) 37 V21: DNA Cloning Restriction Enzymes Cutting DNA at specific sites: restriction endonucleases ↑ Teil des bakterielen "Immunsystems" DNA > exprimieren um Restriction enzymes (restriction endonucleases) are DNAses expressed by - zu schneiden die von bacteria to protect themselves from viruses and other foreign DNA. z B. phagth in Zelle kommen Restriction enzymes recognize specific short DNA sequences in viral genomes, and then cut them up into pieces. nicht Bacteria protect their own genomes through methylation of a base (often A) geschützt % within the recognition sequence. Only unmethylated DNA is cleaved. The fragments generated when a molecule of DNA is digested with a restriction enzyme are known as restriction fragments. O & 38 Eigenschutz der Bakterien O vor restriction enzymes V21: DNA Cloning Restriction Enzymes Cutting DNA at specific sites: restriction endonucleases Restriction endonucleases are very common: all bacteria and archaea appear to have them. There are three general types of restriction endonucleases. Virtually all recombinant DNA work is done with type II enzymes. Type II restriction enzymes recognize short (4-8 bp), usually palindromic (same sequence on both strands) sequences, and cut both strands within or very close to that recognition site, leaving a 5’-phosphate and a 3’-OH. There are now over 3’000 characterized type II restriction endonucleases, with specificities for over 250 different sequences. Restriction enzymes that recognize the same -von DNA sequence and cut at the same position are known as isoschizomers (if they recognize the same ( sequence but cut at different positions, they are known as neoisoschizomers). 39 einer schneiden DNA innerhalb Erkehnung sequenz V21: DNA Cloning Restriction Enzymes Examples of type II restriction endonucleases fast immer palintromisch wo schneidet restriktions D enzym 5 O nummer -Reihenfolge der Isolierung P Genus spezies O 40 3-4 letters  (kein Prüfungsstoff) V21: DNA Cloning Restriction Enzymes Nucleotide nomenclature A, C, G, T (U) Examples: W: A or T (U) (Weak H bonds) Acs I S: C or G (Strong H bonds) R A A T T Y M: A or C (aMino group) Y T T A A R K: G or T (U) (Keto group) R: A or G (puRine) BsaB I Y: C or T (U) (pYrimidine) G A T N N N N A T C B: Not A (B follows A) C T A N N N N T A G D: Not C (D follows C) H: Not G (H follows G) V: Not T (U) (V follows T & U) N: (any Nucleotide) R↑AATTY / YTTAA↓R 41 V21: DNA Cloning Restriction Enzymes How frequent are restriction sites? Most type II restriction enzymes recognize 4-8 bp sequences. Short recognition sequences (e.g., GGCC) will be more common than long ones (e.g., GCGGCCGC). Statistical frequency will also depend on the GC content of the DNA. Example: What is the average restriction fragment length of human genomic DNA (40% G/C) digested with the enzyme EcoR I (GAATTC)? 20 % C th - 20 % G ** = 3000 30 % T 30 % A 42  V21: DNA Cloning Restriction Enzymes Cutting of DNA by type II restriction enzymes can leave different kinds of fragment ends Restriction enzymes usually cut symmetrically on both strands Depending on where in the recognition sequence the restriction enzyme cuts, three possible types of ends can be generated: – Blunt ends – 5’ overhangs - S Pst I 4-base 5’ overhangs – 3’ overhangs 5’ N N C T G C A G N N 3’ 3’ N N G A C G T C N N 5’ 00- 5’ N N C T G C A-OH 3’ 5’ PO4-G N N 3’ restriction > - - 3’ N N G-PO4 5’ 3’ HO-A C G T C N N 5’ O fragment 4-base 3’ overhangs 43 kein 51/3'Ende overhangs = sticky ends V21: DNA Cloning Compatible cohesive ends allow restriction fragments to Restriction Enzymes hybridize with each other to form recombinant DNA molecules Kompatible 19leichenüberhang e Jea zum Vektor 5’ and 3’ overhangs are known as cohesive ends or Enden - “sticky ends”, because they can base pair with complementary sequences. T Sticky ends that can base pair with each other are said to ~ have compatible ends. 5'overhang Once the two ends have base paired with each other, DNA ligase can seal the nick in the sugar-phosphate backbone, basen paaring O creating a covalent bond between the two ends. Mensch & Two DNA molecules from different origins that have compatible (complementary) ends can in this way to joined / to generate a new recombinant DNA molecule. Phosphodiether-Verbinding Balaterie(plasmial Gen in Vektor eingebaut erfolgreich Note that blunt ends can also be fused together by DNA wieder ausgebildet ligase, albeit very inefficiently. Blunt ends are thus also erfolgreich Insulin in 44 compatible with each other. Velator ein- gebaut V21: DNA Cloning Ligation and Selection Donor DNA digested with restriction enzymes can be ligated into vectors to generate a DNA library a) Both host vector and donor DNA are cut with enzymes that create compatible ends (usually the same to enzyme). go go o b) The digested vector and donor DNA are mixed and d d allowed to hybridize in the presence of DNA ligase. c) A fraction of donor DNA restriction fragments will sticys hybridize with the vector DNA, generating recombinant molecules that now contain the original vector DNA and a Insertion small amount of donor DNA. When the original donor DNA is of high complexity, Ocomplimentary base pairing the result of this process is a DNA library: a (mit representative collection of recombinant molecules that contain various fragments of the donor DNA. Genom 45 komplettem mit sehr vielen Bruchstücken ganzes Genom gespeichert V21: DNA Cloning Ligation and Selection Possible results of mixing DNA molecules together In a complex mixture, many different types of molecules can be created. a. Circularization of the vector b. Circularization of a donor fragment c. Joining of two vectors together d. Insertion of a single donor DNA fragment into the vector e. Insertion of multiple donor DNA fragments vektor + f. Many others! mehrere Stücke donor DNA der DNA verbunden O 00 O O O Sticky ends des 2 vektoren O Plasmids verbunden zusammengelagert 46 Result: many different recombinant molecules, and only one copy of each of them! sehr hohe lokale Konzentration V21: DNA Cloning Ligation and Selection How to selectively get the recombinant molecule that you want? 1. Selective amplification of only a subset of recombinant molecules (e.g., those that contain at least one copy of vector DNA) 2. Analysis of amplified recombinant molecules to identify the one of interest. 47 V21: DNA Cloning Ligation and Selection Recombinant molecules can be introduced into E. coli in Velter Following ligation, the einfügen netymes ~ verdauen mixture of new recombinant DNA molecules can be introduced into E. coli. 0 Each cell will usually take rekombinantes up at most one Molekül recombinant molecule ↓ in Zelle einfügen f transformation (most will take up none) through a process known as transformation. 48 V21: DNA Cloning Ligation and Selection Recombinant molecules can be amplified in E. coli Each bacterial cell carrying a new recombinant molecule will now grow, creating a group of identical cells (group of clones), all carrying the same relaxed plasmid recombinant molecule. Each bacterial clone will thus carry one or S more copies of one particular recombinant C molecule. Following amplification (bacterial growth), wu the recombinant DNA molecule can easily r gleichen be purified out of the bacteria. Vektor + Stück ein- The use of recombinant DNA to generate gebautes DNA many copies of a recombinant DNA molecule is known as cloning. 49 L Clone plasmide ⑦ Anzahl vektoren können einfach den Bakterien => bacteria growth aus isoliert werden V21: DNA Cloning Phage Vectors Cloning vectors for very large DNA fragments koin - im ↳ Synthetische Plasmide Phage P1 artificial chromosomes (PACs) are derivatives of the bacteriophage P1, and can contain 80-100 kb of donor DNA. Bacterial artificial chromosomes (BACs) are basically very large plasmids, that can contain up to 300 kb of donor DNA. Yeast artificial chromosomes (YACs) are, as their name implies, artificial chromosomes that can be replicated in the yeast S. cerevisiae. They can contain over 50 a Mb of donor DNA. Megabasen  nicht essentielle Gehe DNADNA V21: Cloning: Cloning haben PhagePhage Vectors Vectors Bacteriophage vectors Most commonly used bacteriophage vectors are based on phage l (lamda). O The middle third of the lamda genome is not essential entfachen for replication, and can be replaced by foreign DNA. ↳ Recombinant phage genomes can be packaged into viral heads in vitro, and used to infect bacteria. O Infection of bacteria on a lawn creates plaques, from Gehe of which the recombinant phage can be isolated and interest purified. einfügen 0000 Phage mit of interest gene 51 Agar-platte plaques : ↓ Region no phage amplifizieren? vervielfältigt haben V21: DNA Cloning Vectors DNA cloning in a plasmid vector /De insulin g.. 1. Restriction digestion of plasmid vector and donor DNA reisane es 2. Ligation of donor DNA and plasmid vector DNA to generate a recombinant DNA molecule transformation 3. Transformation of ligation mix into host cells (E. coli) - 4. Distribution of transformed bacteria onto agar plates X 5. Amplification of plasmid in host cells 6. Multiplication of transformed bacteria into bacterial colonies O Question: why do E. coli cells need to be distributed on agar plates following transformation? - -O S 52 sehr hohe Anzahl des Vektors V21: DNA Cloning Vectors Video “DNA cloning” 53 V21: DNA Cloning Ligation and Selection Possible results of mixing DNA molecules together In a complex mixture, many different types of molecules can be created. a. Circularization of the vector b. Circularization of a host fragment c. Joining of two vectors together d. Insertion of a single donor DNA fragment into the vector e. Insertion of multiple donor DNA fragments f. Many others! 54 Result: many different recombinant molecules, and only one copy of each of them! V21: DNA Cloning Ligation and Selection Identification of plasmids containing an insert 55 V21: DNA Cloning Ligation and Selection The blue-white selection can identify plasmid vectors that contain an insert site multiple clowing restriction enzyme fo Many vectors, such as pBluescript, contain a lacZ’ gene, which, to lac Z Kaphit in the right bacterial background, allows for the production of a B-gay - functional LacZ (b-galactosidase) enzyme. DNA -Donor Insertion of a foreign DNA fragment into the MCS located keine B- galactosidase within the lacZ’ gene usually inactivates the lacZ’ gene. mehr Active b-galactosidase can cleave the chromogenic substrate X-gal into a blue product. kam Farbstoff im Nährboden spalten blauer Farbstoff Bacteria that have taken up the original plasmid vector make blue colonies; colonies from bacteria that have taken up a plasmid containing an insert (foreign DNA) are white. colonies blue Question: Why does insertion of a donor DNA fragment into a restriction site within lacZ’ usually inactive the gene? white colorient 0 O 56 V21: DNA Cloning Ligation and Selection White colony lac Z Kaputt ; donor DNA eingefügt; ampicilinresistant , not blue > - donor DNA wurde hinzugefügt Blue colony B-glactosidase aktiv => relator ohne DOLOr DNA How can you find out what is inserted into the MCS? 57 V21: DNA Cloning Ligation and Selection Possible results of mixing DNA molecules together In a complex mixture, many different types of molecules can be created. a. Circularization of the vector b. Circularization of a host fragment c. Joining of two vectors together d. Insertion of a single donor DNA fragment into the vector e. Insertion of multiple donor DNA fragments f. Many others! 58 Result: many different recombinant molecules, and only one copy of each of them! V21: DNA Cloning Ligation and Selection Analysis of recombinant DNA molecules - Analysis of insert size(s) – Restriction digestion and gel electrophoresis Analysis of insert sequence (lecture 22) – Hybridization to a probe – DNA sequencing 59 V21: DNA Cloning Analysis of DNA Fragments Restriction digestion of linear and circular molecules Digestion of a recombinant DNA molecule with restriction enzymes will generate restriction fragments. + The number and size of the fragments depend on the sequence and topology of the DNA molecule. – Digestion of a linear molecule with n restriction sites generates n+1 fragments. – Digestion of a circular molecule with n restriction sites generates n fragments. 60 V21: DNA Cloning Gel electrophoresis separates DNA molecules based Analysis of DNA Fragments on their size Electrophoresis uses an electric field to separate molecules based on their charge, size and shape. er a Migration is normally done in a mesh-like matrix, either of agarose (for DNA > 100 bp) or of polyacrylamide (for DNA < 1000 bp). ↓ synthetic polymer DNAauftragen & For molecules with uniform charge density and similar shapes (such as DNA), migration in an electric field -is DNA ' Kindurch directly related (inversely proportional) muss wandern to the log of the size of the molecule. V kleine Moleküle - wandern schnell - weit unten ⑦ DNA sichtbar Wanderungsgeschwindigkeit 61 V21: DNA Cloning Analysis of DNA Fragments Agarose gel electrophoresis allows the separation and visualization of DNA molecules insert - ↓ O 00000 62 I Vektor grössenstandart ein gleicher Grössel V21: DNA Cloning Analysis of DNA Fragments Separated DNA molecules can be visualized though the use of fluorescent DNA intercalators DNA fragments can be detected through the use of fluorescent dyes that specifically bind to dsDNA (e.g., ethidium bromide). Ethidium bromide can intercalate between DNA base pairs. Once intercalated, ethidium bromide fluoresces very strongly. schieben sich zwischen stacked base pairs hinein ALL DNA molecules present in the gel will bind the dye and fluoresce. Fluorescence intensity will depend on size and amount of DNA present: as little as 10 ng of DNA can be detected using - this approach. Other fluorescent molecules will detect even lower amounts of DNA. ethidium bromide Question: how many molecules of DNA does 10 ng correspond to? Assume a 1 kb fragment. 1 base pair = 660 g/mol; NA: 6.02 x 1023 molecules/mol 63 V21: DNA Cloning Analysis of DNA Fragments Size markers allow the estimation restriction fragment sizes start (wells) big fragments direction of migration ~ Darau ↑ small fragments 64 für Bande jeder Wieviel Basenpaarung entspricht  of intererst gehe amplifizieren/identifizieren V21: DNA Cloning PCR How to amplify a DNA fragment of interest The problem: genes are minute, and there are thousands of them in every cell. How do you get enough DNA of the gene you care about to actually be able to work with it? Solution: selective amplification of your favorite gene (often abbreviated yfg-1) Two general approaches for amplifying (making many copies) of a DNA molecule: a. Cloning b. Polymerase chain reaction (PCR) 65 V21: DNA Cloning PCR In vitro amplification of DNA fragments: the polymerase chain reaction (PCR) PCR is basically in vitro DNA replication; it will thus require a single-stranded DNA template, a primer, a DNA polymerase, and dNTPs. stranded DNA single - primer - - DNA polymerace - activated nucleotides 66 V21: DNA Cloning PCR In vitro amplification of DNA fragments: - M Heart the polymerase chain reaction (PCR) & -0 PCR is basically in vitro DNA replication; it will thus require a single-stranded DNA template, a primer, a DNA polymerase, synthetisieren DNAsynthese and dNTPs. Komplimentärerstag & Hear Because the reaction occurs in vitro, there are also a few ↓ differences in how we solve some of the challenges: gekühlt 1. Strand separation is achieved through heat, not through helicases. à The DNA polymerase must thus be able to withstand high temperatures. All PCR polymerases come from bacteria that live in very hot places (hot springs, ocean vents). 67 V21: DNA Cloning PCR DNA polymerases for PCR The DNA polymerases used in PCR come from bacterial species that live in very hot places Hot spring in Yellowstone National Park, USA – the home of Thermus 68 aquaticus, the source of Taq DNA polymerase. V21: DNA Cloning In vitro amplification of DNA fragments: PCR the polymerase chain reaction (PCR) PCR is basically in vitro DNA replication; it will thus require a single- stranded DNA template, a primer, a DNA polymerase, and dNTPs. Because the reaction occurs in vitro, there are also a few differences in how we solve some of the challenges: 1. Strand separation is achieved through heat, not through helicases. à The DNA polymerase must thus be able to withstand high temperatures. All PCR polymerases come from bacteria that live in very hot places (hot springs, ocean vents). 2. In PCR, the molecular biologist adds specific primers to the reaction, rather than letting DNA primase synthesize primers everywhere. à Thus, only a specific segment of DNA will be replicated. 69 V21: DNA Cloning PCR PCR allows an exponential amplification of target DNA PCR consists of many (usually 25-35) cycles of DNA replication. In each cycle, only the region of interest is replicated, as only the oligonucleotides added by the biologist can be used as primers by the DNA polymerase. If a set of two primers are chosen close enough to each other, the region between the two primers can be amplified exponentially. 70 V21: DNA Cloning PCR PCR is amazingly powerful Using PCR, you can amplify a DNA fragment starting O with as little as 1 cell! à Useful for forensic analysis, prenatal diagnosis, biopsy analysis, etc. Using PCR, DNA has been amplified (and then cloned) from Egyptian mummies, from Oetzi, even from Neanderthal bones!!! This also shows how stable DNA is. However, no DNA has ever been amplified success- The Nobel Prize in Physiology or fully from Dinosaurs (Jurassic Park is bogus!) Medicine 2022 Note that because PCR is so sensitive, you also “for his discoveries concerning the have to be very careful about not contaminating your genomes of extinct hominins and human DNA sample! evolution” 71 Take-home messages Restriction enzymes can cleave DNA at specific, usually palindromic sequences, to generate restriction fragments. The ability of compatible restriction fragments to hybridize to each other allows the generation of recombinant DNA molecules. Recombinant DNA molecules can be amplified in hosts. This is known as DNA cloning. Many different vectors have been developed to allow the cloning of DNAs of different sizes, and to allow expression of proteins encoded by the donor DNA. DNA fragments can be separated based on size by gel electrophoresis. Polymerase chain reaction (PCR) can be used to exponentially amplify small DNA fragments in vitro. PCR is extremely powerful and sensitive (very little starting material is required). 72  Keywords (Schlüsselwörter) Recombinant DNA (Rekombinante DNS) Genetic engineering (Gentechnologie) Restriction enzyme (Restriktionsenzym) Restriction fragment (Restriktionsfragment) Palindrome (Palindrom) Blunt ends (stumpfe Enden) Cohesive ends (cohesive Enden) 5’ overhang (5’ Überhang) 3’ overhang (3’ Überhang) Clone (Klon) To clone (klonieren) Vector (Vektor) Plasmid (Plasmid) Cosmid (Cosmid) 73