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Recombinant DNA Technology Recombinant DNA Technology • The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973 • They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro” – Which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium • Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith – For which they received the 1978 Nobel Prize in Medicine Recombinant DNA technology – a set of techniques for combining genes from different sources • recombinant DNA,, is made by connecting or recombining, fragments of DNA from different sources ( often different species ) • These methods form part of genetic engineering, the direct manipulation of genes for practical purposes (Process of altering the genetic material of cells or organisms to make new substances) • DNA technology has launched a revolution in biotechnology, the manipulation of organisms or their components to make useful products Uses for Recombinant DNA Recombinant DNA technology is not only an important tool in scientific research, but it has also impacted the diagnosis and treatment of diseases and genetic disorders in many areas of medicine. It has enabled many advances, including: • Isolation of large quantities of pure protein • Diagnosis of affected and carrier states for hereditary diseases • Transferring of genes from one organism to another • Mapping of human genes on chromosomes • Identification of mutations – People may be tested for the presence of mutated proteins that may be associated with breast cancer, retino-blastoma, and neurofibromatosis Tools & Techniques of genetic engineering • enzymes for dicing, splicing, & reversing nucleic acids • analysis of DNA 5 Enzymes for dicing, splicing, & reversing nucleic acids 1. restriction endonucleases – recognize specific sequences of DNA & break phosphodiester bonds 2. ligase – rejoins phosphate-sugar bonds cut by endonucleases 3. reverse transcriptase – makes a DNA copy of RNA - cDNA 6 Some enzymes used in recombinant DNA technology Analysis of DNA • gel electrophoresis- separates DNA fragments based on size • nucleic acid hybridization & probes – probes base pair with complementary sequences; used to detect specific sequences • DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA • Polymerase Chain Reaction – way to amplify DNA 8 DNA Amplification • DNA amplification is based on two different techniques : a- Cell-based DNA cloning(DNA cloning) involving a vector/replicon and a suitable host cell . b- in vitro DNA cloning (PCR) DNA Cloning (cell-based cloning) The goal of molecular cloning is large amounts of pure DNA that can be further manipulated and studied. A clone is an identical copy. This term originally applied to cells of a single type, isolated and allowed to reproduce to create a population of identical cells. • Principles of cell-based cloning: Four steps in cell-based cloning - Construction of recombinant DNA molecules. Involves the use of endonuclease restriction enzymes, ligation, and a replicon (vector). - Transformation in appropriate host cells. - Selective propagation of cell clones. This step takes advantage of selectable markers. - Isolation of recombinant DNA from cell clones followed by molecular characterization . Major Steps in the Cloning of DNA • Fragmentation of DNA : – Appropriate restriction endonucleases is used to ensure that the gene fragment in question is excised completely from the source of DNA . • Construction of recombinant DNA ( rDNA)molecule : • · The target gene fragment is ligated to a DNA vector (i.e. a plasmid), making a recombinant DNA molecule • · The DNA recombinant molecule replicates itself autonomously • Transformation of recombinant DNA into the host cell . • Cell cloning . Major Steps in the Cloning of DNA • The selection of the successfully transformed cells -Isolation of successfully transformed bacterial cells with the recombinant DNA using a marker, such as antibiotic resistance genes -If colonies grow, despite the existence of such antibiotics, then the recombinant DNA vector was successfully transformed • The surviving colonies are isolated and are grown in culture to produce multiple copies of the incorporated recombinant DNA • Isolation of recombinant DNA from cell clones followed by molecular characterization Fragmentation of DNA Restriction Enzymes • Restriction enzymes are Bacterial origin = enzymes that cleave foreign DNA • classified as endonucleases. Their biochemical activity is the hydrolysis ("digestion") of the phosphodiester backbone at specific sites in a DNA sequence. By "specific" it means that an enzyme will only digest a DNA molecule after locating a particular sequence. • All restriction enzymes cut DNA between the 3’ carbon and the phosphate moiety of the phosphodiester bond. • The term restriction comes from the fact that these enzymes were discovered in E. coli strains that appeared to be restricting the infection by certain bacteriophages • Over 400 enzymes identified, many available commercially from biotechnology companies • Names typically begin with 3 italicized letters ex. EcoRI source E. coli RY13 , BamHI source Bacillus amyloliquefaciens H Origin and function • Bacterial origin = enzymes that cleave foreign DNA • Protect bacteria from bacteriophage infection – Restricts viral replication – However, certain bacteriophages have evolved to use methylation as a way to avoid digestion by restriction enzymes • Bacterium protects it’s own DNA by methylating those specific sequence motifs Restriction Enzymes • Restriction enzymes bind to, recognize, and cut (digest) DNA within specific sequences of bases called a recognition sequence or restriction site • These recognition sequences are palindromes ( arrangement a of nucleotides reads the same forwards and backwards on opposite strands of the DNA molecule ) • They typically recognize restriction sites with a sequence of four or six nucleotides Eight-base pair cutters have also been identified • They produce either Blunt Ends or Staggered ends: Staggered ends Blunt Ends Staggered ends Blunt Ends Restriction Enzymes • Enzymes that produce cohesive ends are often favored over blunt-end cutters for many cloning experiments • But blunt ends can be converted to sticky ends by linkers . – DNA fragments with cohesive ends can easily be joined together . • In the simplest sense, the discovery of restriction enzymes provided molecular biologists with the "scissors" needed to carry out gene cloning linkers • Blunt ends can be converted to sticky ends. • A short double-stranded molecules called linkers or adaptors are attached to the blunt ends. Linkers and adaptors work in slightly different ways but both contain a recognition sequence for a restriction endonuclease and so produce a sticky end after treatment with the appropriate enzyme LINKER Another way to create a sticky end • Homopolymer tailing - Nucleotides are added one after the other to the 3′ terminus at a blunt end . • The enzyme involved is called terminal deoxynucleotidyl transferase . End-modification enzymes • Terminal deoxynucleotidyl transferase from calf thymus tissue, is one example of an end-modification enzyme. • It is a template-independent DNA polymerase because it is able to synthesize a new DNA polynucleotide without base-pairing of the incoming nucleotides to an existing strand of DNA or RNA. Its main role in recombinant DNA technology is in homopolymer tailing • Two other end-modification enzymes are also frequently used , alkaline phosphatase and T4 polynucleotide . Restriction Enzymes • site-specific endonucleases of prokaryotes • function to protect bacteria from phage (virus) infection • corresponding site-specific modifying enzyme (eg., methylase) • type II enzymes are powerful tools in molecular biology Classes of Restriction Enzymes cleavage occurs 400-7000 bp Type I from recognition site cleavage occurs adjacent or Type II within recognition site cleavage occurs 25-27 bp Type III from recognition site Classes • Type I • – Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme – Therefore random/imprecise cuts – Not very useful for rDNA applications Type III (similar to type I , few examples ) - Recognition sequence: 5-7 bp - Cleavage site: 25-27 bp downstream of recognition site • Type II – Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme – Used widely for molecular biology procedures – DNA sequence = symmetrical • Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence Some Examples of Restriction Endonucleases Vectors • A vector is a DNA molecule into which foreign fragments of DNA is inserted • A vector functions like a “molecular carrier” – Which will carry fragments of DNA into a host cell – Vector DNA functions to insert and amplify the DNA of intersit . • Vectors should contain an origin of replication – Enables the vector, together with the foreign DNA fragment inserted into it, to replicate • they contain one or more single (unique) restriction endonuclease sites that provide a choice of possible insertion (cloning) sites • vectors have one or more genes (selectable markers) that enable host cells with DNA constructs to be distinguished from cells that either do not carry a DNA construct or carry a cloning vector without an insert. • Small size in comparison with host’s chromosomes Main types of vectors Choice of vector • Depends on nature of protocol or experiment • Length of the DNA molecule . • Type of host cell to accommodate rDNA – Eukaryotic - Prokaryotic • Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals. Plasmid vector • Closed, circular, double stranded DNA molecules that occur naturally and replicate extrachromosomally in bacteria • a multiple cloning site or MCS • Many confer drug resistance to bacterial strains • Origin of replication present (ORI) • pBR322 is the basis of most engineered plasmids Plasmid vector Another example of a typical E. coli cloning vector is pUC19 (2,686-bp). The pUC19 plasmid features: a. High copy number in E. coli, with nearly a hundred copies per cell, provides a good yield of cloned DNA. b. Its selectable marker is ampR. c. It has a cluster of unique restriction sites, called the polylinker (multiple cloning site). d. The polylinker is part of the lacZ (β-galactosidase) gene. The pUC19 plasmid will complement a lacZ- E. coli, allowing it to become lacZ+. When DNA is cloned into the polylinker, lacZ is disrupted, preventing complementation from occurring. e. X-gal, a chromogenic analog of lactose, turns blue whenβgalactosidase is present, and remains white in its absence, so bluewhite screening can indicate which colonies contain recombinant plasmids The plasmid cloning vector pUC19 Chapter 7 slide 32 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Plasmid vector • Artificial plasmids have been created that possess a unique region that can be cut by many restriction enzymes • The recognition sites of many restriction enzymes have been positioned very close together in this one area and are not found anywhere else on the plasmid’s DNA sequence – the site is called the multiple-cloning site • The recognition site exists in only one area of the plasmid which means that the DNA can only be cut at one location Bacteriophage Vectors Lambda vector • Bacteriophage lambda (λ) infects E. coli • Double-stranded, linear DNA vector – suitable for library construction • At each end of the λ chromosome are 12 nucleotide sequences called cohesive sites (COS) that can base pair with each other - When λ infects E. coli as a host, the λ chromosome uses these COS sites to circularize and then replicate • Can accommodate large segments of foreign DNA • Central 1/3 = “stuffer” fragment – Can be substituted with any DNA fragment of similar size without affecting ability of lambda to package itself and infect E. coli – Accommodates ~15kbp of foreign DNA – Foreign DNA is ligated to Left and Right Arms of lambda Then either: • 1) Transfected into E. coli as naked DNA, or • 2) Packaged in vitro by combining with phage protein components (heads and tails) (more efficient, but labor intensive and expensive) • As λ replicates to create more viral particles, infected E. coli are lysed by λ using phage λ DNA as a cloning vector Chapter 7 slide 36 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Cosmid cloning vectors 1. Cosmids are constructed vector with features from both plasmids and phages. These features include: a. b. c. d. An E. coli ori sequence. A selectable marker such as ampR Convenient restriction sites. A phage cos site, allowing the DNA to be pakaged into a phage head for introduction into E. coli. 2. Cosmid as small as 5kb are available, and 32-47kb of DNA can be inserted into them. Recombinant cosmids < 37kb or > 52 kb cannot be packaged, don’t enter E. coli, and therefore are not replicated. Cosmid cloning vector Yeast Artificial Chromosomes (YACs) • Bacterial plasmids are not good vectors for yeast hosts because prokaryotic and eukaryotic DNA sequences use different origins of replication. • A yeast artificial chromosome, or YAC, has been made that has a yeast origin of replication, a centromere sequence, and telomeres, making it a true eukaryotic chromosome. • YACs have been engineered to include specialized single restriction sites and selectable markers. • YACs can accommodate up to 1.5 million base pairs of inserted DNA. Yeast Artificial Chromosome Example of a yeast artificial chromosome (YAC) cloning vector. A YAC vector contains a yeast telomere (TEL) at each end, a yeast centromere sequence (CEN), a yeast selectable marker for each arm (here, TRP1 and URA3), a sequence that allows autonomous replication in yeast (ARS), and restriction sites for cloning. Bacterial Artificial Chromosomes (BACs) 1. BACs are used for cloning fragments up to about 200 kb in E .coli. BAC vectors contain: a. the ori of an E. coli plasmid called the F factor. b. A multiple cloning sites. c. A selectable marker. d. Other features 2. BAC can be handles like regular bacterial plasmids, but the F factor ori keeps copy number at one BAC molecule per cell. Expression Vectors • Protein expression vectors allow for the high -level synthesis (expression) of eukaryotic proteins within bacterial cells – They contain a prokaryotic promoter sequence adjacent to the site where DNA is inserted into the plasmid • Bacterial RNA polymerase can bind to the promoter and synthesize large amounts of RNA (for the insert) – Which is then translated into protein • Protein may then be isolated using biochemical techniques Ti Vectors • Ti vectors are naturally occurring plasmids (around 200 kb in size) – Isolated from the bacterium Agrobaderium tumefaciens • Which is a soilborne plant pathogen that causes a condition in plants called crown gall disease • When A. tumefaciens enters host plants, a piece of DNA (T-DNA) from the Ti plasmid (Ti stands for tumor-inducing) inserts into the host chromosome Ti Vectors • T-DNA encodes for the synthesis of a hormone called auxin, which weakens the host cell wall – Infected plant cells divide and enlarge to form a tumor (gall) • Plant geneticists recognized that if they could remove auxin and other detrimental genes from the Ti plasmid, the resulting vector could be used to deliver genes into plant cells • Ti vectors are widely used to transfer genes into plants Shuttle Vectors 1.A cloning vector capable of replicating in two or more types of organism (e.g., E. coli and yeast) is called a shuttle vector. Shuttle vectors may replicate autonomously in both hosts, or integrate into the host genome Construction of recombinant DNA ( rDNA)molecule • As the DNA of interest and the vector are digested by the same restriction enzyme ,they have a complementary ends . • The target DNA fragment is ligated to a DNA vector (i.e. a plasmid) by ligase enzyme, making a recombinant DNA molecule Transformation • Recombinant DNA molecules are transferred into appropriate host cells (e.g. bacteria) for propagation. Normally a single recombinant DNA exists per cell but sometimes cotransformation may result in two or more recombinant DNA molecules per host cell. Characteristics of cloning hosts 1. Rapid overturn, fast growth rate 2. Can be grown in large quantities using ordinary culture methods 3. Nonpathogenic 4. Capable of accepting plasmid or bacteriophage vectors 5. Maintains foreign genes through multiple generations 6. Will secrete a high yield of proteins from expressed foreign genes 48 Transformation • Transformation – Process for inserting foreign DNA into bacteria – the bacteria that has accepted a foreign plasmid is referred to as being transformed . • I Cells (generally E. coli) and plasmid DNA are incubated together at 0 C in a calcium chloride solution then the mixture is subjected to a shock by rapidly shifting the temperature to 43 C. Result : Plasmid DNA entered bacterial cells • Once inside bacteria, plasmids replicate and express their genes Transformation • II Electroporation : – Involves applying a brief (millisecond) pulse of high-voltage electricity to create tiny holes in the bacterial cell wall that allow DNA to enter – Can be used to introduce DNA into mammalian cells and to transform plant cells • Ligation of DNA fragments and transformation by any method are some what inefficient • During ligation, some of the digested plasmid will ligate back to itself to create a recircularized plasmid that lacks foreign DNA • During transformation, a majority of cells will not take up DNA Cloning of DNA: technique pBR322 52 Antibiotic Selection • How can recombinant bacteria (those transformed with a recombinant plasmid) be distinguished from a large number of non transformed bacteria and bacterial cells that contain plasmid DNA without foreign DNA? – Screening process is called selection • Designed to facilitate the identification of (selecting for) recombinant bacteria • Example "blue blue-white" screening by using the plasmid cloning vector pUC19 . ( Fig ) Practical Features of DNA Cloning Vectors • Transformed bacteria are plated on agar plates that contain a chromogenic (colorproducing) substrate for β-gal called X-gal – (5-bromo-4-chloro-3-indolylβ-[5]Dgalactopyranoside) • X-gal is similar to lactose in structure and turns blue when cleaved by β-gal • As a result, non-recombinant bacteria, which contain a func-tionallac z gene, produce β-gal and turn blue • Conversely, recombinant bacteria are identified as white colonies • Because these cells contain plasmid with foreign DNA inserted into the lac z gene, β-gal is not produced, and these cells cannot metabolize X-gal. Recombinant selection with pBR322 Recombinant selection with pUC8 . •